Methods and compositions for modulating toso activity

ABSTRACT

The present invention is further directed to methods and compositions for modulating the activity of the Toso protein. The invention further encompasses treatment of disorders associated with inflammation, autoimmune disorders, and cancer using compositions that include a soluble Toso protein.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. application Ser. No.13/831,031, filed Mar. 14, 2013, which claims the benefit of priority toU.S. Provisional Application No. 61/612,183, filed Mar. 16, 2012, U.S.Provisional Application No. 61/646,143, filed May 11, 2012, and U.S.Provisional Application No. 61/731,428, filed Nov. 29, 2012, thecontents of which are expressly incorporated herein by reference intheir entirety for all purposes.

BACKGROUND OF THE INVENTION

Toso or Faim3 (Fas Apoptotic inducing molecule 3) is a single membranespanning cell surface receptor originally characterized through aretroviral overexpression screen in Jurkat cells, a T cell leukemicline, as a mediator of Fas-induced apoptotic cell death (Hitoshi, Y., etal., Toso, a cell surface, specific regulator of Fas-induced apoptosisin T cells. Immunity, 1998. 8(4): p. 461-71). Subsequent studies havesuggested that Toso is the elusive receptor for IgM. The expression ofToso also seems to correlate with particularly aggressive forms ofChronic Lymphocytic Leukemia, or CLL.

There is a need for characterization of the in vivo role of Toso inorder to identify its use as a therapeutic target and for compositionscomprising agents that can bind to Toso and/or modulate its activity.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides methods and compositions formodulating Toso activity and treating diseases and disorders in whichToso is implicated.

In one aspect, the present invention provides a method of treating anautoimmune disorder in a subject, the method including a step oftreating the subject with a composition containing a therapeuticallyeffective amount of a soluble Toso protein. In exemplary embodiments,the autoimmune disorder is without limitation rheumatoid arthritis,multiple sclerosis, lupus or Type I diabetes.

In a further aspect, the present invention provides a method of treatingType II diabetes in a subject, the method including a step of treatingthe subject with a composition comprising a therapeutically effectiveamount of a soluble Toso protein.

In one aspect, the present invention provides a method of treatingasthma in a subject, the method including a step of treating the subjectwith a composition comprising a therapeutically effective amount of asoluble Toso protein.

In a further aspect, the present invention provides methods of treatingdiabetes, asthma, multiple sclerosis, or rheumatoid arthritis in asubject by administering a soluble polypeptide having an amino acidsequence of SEQ ID NO: 5 or 6 or a fragment or a deletion variantthereof to that subject. In further embodiments, the Toso activity inthe subject is reduced in the subject. In still further embodiments, thepresent invention provides methods of treating diabetes, asthma,multiple sclerosis, or rheumatoid arthritis in a subject byadministering a soluble polypeptide comprising an amino acid sequence ofany one or more of SEQ ID NOs. 1-25.

In a further aspect, the present invention provides methods of treatingcancer, allergy, COPD, hyper-IgM syndrome, lupus, or aneutrophilia-associated disorder in a subject, the method including astep of treating the subject with a composition comprising atherapeutically effective amount of a soluble Toso protein.

In further embodiments and in accordance with any of the above, thesoluble Toso protein of the invention includes amino acid residues aminoacids P21 to G251 of NP_(—)005440.1, human Toso isoform a (see Shima etal., Int. Immunol., 2010, which is hereby incorporated in its entiretyfor all purposes and in particular for all teachings related to theextracellular domain of Toso).

In still further embodiments and in accordance with any of the above,the soluble Toso protein of the invention includes the extracellulardomain of a human Toso protein. In further embodiments, the soluble Tosoprotein of use in the invention includes amino acid residues 18-253 ofSEQ ID NO: 7.

In still further embodiments and in accordance with any of the above,the soluble Toso protein used in methods of the present inventioncomprises an amino acid sequence according to SEQ ID NO: 5 or 6. In yetfurther embodiments, the soluble Toso protein comprises an amino acidsequence with at least 90% sequence identity to SEQ ID NO: 5 or 6. Inyet further embodiments, the soluble Toso protein of the presentinvention includes deletion variants of SEQ ID NO: 5 or 6. In stillfurther embodiments and in accordance with any of the above, the solubleToso protein used in methods of the present invention comprises an aminoacid sequence of any one or more of SEQ ID NOs. 1-25.

In further embodiments and in accordance with any of the above, thesoluble Toso protein of the invention is a multimer. In still furtherembodiments, the multimer is made up of 6 monomers. In yet furtherembodiments, each monomer of the multimeric Toso protein comprises asequence according to SEQ ID NO: 6.

In a further aspect, the present invention provides methods forinhibiting Toso activity that include applying a soluble Tosopolypeptide to a cell comprising a membrane-bound Toso receptor.

In a still further aspect, the present invention provides a compositionthat includes a polypeptide of SEQ ID NO: 5 or SEQ ID NO: 6. In oneembodiment, that composition inhibits Toso activity. In a furtherembodiment, the polypeptide is a multimer. In a still furtherembodiment, the multimer includes 6 monomers, where each monomercomprises a sequence according to SEQ ID NO: 6.

In a further embodiment, the present invention provides an isolatednucleic acid encoding a soluble Toso protein in accordance with any ofthe protein described herein. In a still further embodiment, the presentinvention provides a host cell expressing an isolated nucleic acidencoding a soluble Toso protein in accordance with any of the proteindescribed herein.

In a further aspect, the present invention provides a fusion proteincomprising an extracellular domain of a human Toso protein, an Fcregion, and a multimerization tag. In a further embodiment, theextracellular domain comprises amino acids 21-251 of a human Tosoprotein. In a still further embodiment, the multimerization tagcomprises SEQ ID NO: 4. In a yet further embodiment, the Fc regioncomprises SEQ ID NO: 3.

In a further embodiment, the present invention provides an isolatednucleic acid encoding the fusion protein described above. In a stillfurther embodiment, the present invention provides a host cellcomprising a nucleic acid encoding the fusion protein described above.

In a further aspect, the present invention provides an isolated, solublepolypeptide comprising an amino acid sequence having at least 90%sequence identity to SEQ ID NOs. 8-24 or to a polypeptide regioncomprising amino acid residues 18-251 of SEQ ID NO: 7.

In a further aspect, the present invention provides methods forproducing a polypeptide encoding any of the soluble Toso proteinsdescribed herein, the method including providing a cell comprising anucleic acid encoding said polypeptide, the cell is cultured underconditions suitable for expression of said polypeptide. In furtherembodiments, the present invention provides a nucleic acid encoding anyof the soluble Toso proteins described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, FIG. 1B and FIG. 1C show data related to inflammation inToso^(−/−) mice. FIG. 1A shows measures of joint inflammation asmeasured by the change in ankle thickness using a digital caliper. FIG.1B shows disease severity scores for each joint based on visibleswelling and mobility. FIG. 1C shows flow cytometric analysis oflymphocyte populations in the draining lymph node.

FIG. 2A, FIG. 2B and FIG. 2C show data from an OVA induced asthma modelon Toso^(−/−) and wild type mice. FIG. 2A is a schematic illustration ofthe induction of the asthma model. FIG. 2B shows data on eosinophilmigration into the broncheoalveolar space as assessed by DifQuikstaining. FIG. 2C shows data on eosinophils quantified by counting atleast 200 leukocytes per slide.

FIG. 3A, FIG. 3B and FIG. 3C show data on OVA induced T_(H)2 cytokinesIL-4, IL-5 and IL-13 in BALF as assessed by ELISA. FIG. 3D shows data onEotaxin, an eosinophil attracting C—C chemokine, in the BALF. FIG. 3Eshows the levels of Eotaxin produced in response to TNFα treatment insmooth muscle cells.

FIG. 4A, FIG. 4B and FIG. 4C show data on IgE in Toso^(−/−) mice. FIGS.4A and B show total and specific IgE levels in Toso^(−/−) mice, and FIG.4C shows IgG1 levels between the wildtype and Toso^(−/−) mice.

FIG. 5 shows Penh values of Toso^(−/−) mice compared to wildtypecontrols.

FIG. 6 shows EAE scores in a MOG-induced model in wildtype andToso^(−/−) mice.

FIG. 7A shows data on cytokine production in the Broncho Alveolar LavageFluid (BALF) of wildtype animals in response to aerosolled OVA that hadbeen previously intra-tracheally installed with Toso^(−/−) and wildtypedendritic cells. FIG. 7B shows proliferation of T cells derived from 2d2mice cultured with MOG₃₅₋₅₅ loaded Toso^(−/−) dendritic cells. FIG. 7Cshows blood glucose levels in RIP-GP animals injected intravenously withGP loaded Toso^(−/−) and wildtype dendritic cells and a Kaplan-Meierplot of RIP-GP mice injected with GP peptide loaded wildtype andToso^(−/−) dendritic cells. RIP-GP mice are mice expressing the majorglycoprotein (GP) from lymphocytic chorio meningitis virus (LCMV) undercontrol of the rat insulin promoter (RIP)—such mice develop diabetes asassessed by increased levels of serum glucose.

FIG. 8A is a schematic illustration of the Toso soluble receptor. FIG.8B shows ELISA data on the soluble receptor. FIG. 8Ci and FIG. 8Ciishows data confirmation of the secretion of the Toso soluble receptor byWestern Blot (FIG. 8Ci) and by Coomassie Blue staining (FIG. 8Cii). FIG.8D shows binding data of the Toso soluble receptor to splenocytes. FIG.8E shows further data indicating that Toso-Fc bound most significantlyto CD11c+MHChi mature dendritic cells, CD4+ T cells and B220+ B cells inthe spleen. FIG. 8F shows ELISA results of Toso-Fc binding to plateboundhuman IgM. FIG. 8G shows normal weight gain in mouse treated with 50 μgdaily of Toso-Fc.

FIG. 9A is a schematic illustration of the treatment protocol for themurine model of OVA induced asthma. FIG. 9B shows data on Th2 cytokinesin the BALF and FIG. 9C shows cellularity data in the BALF, which areboth measurements of the severity of the disease.

FIG. 10A, FIG. 10B, FIG. 10C and FIG. 10D provide sequences ofembodiments of the invention, including that of a soluble Toso protein(FIG. 10A), an IL-2 signal sequence (FIG. 10B), an Fc domain (FIG. 10C)and a hexamerization tag (FIG. 10D).

FIG. 11A provides a sequence of an embodiment of a soluble Toso proteincomprising a hexameric tag, allowing for expression of a multimeric formof the soluble receptor. FIG. 11B provides a sequence of a human Tosoprotein, NP_(—)005440.1 (SEQ ID NO: 7).

FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D, FIG. 12E, FIG. 12F and FIG. Gshow TOSO expression on granulocytes. FIG. 12A: RNA was isolated fromspleens and livers of wildtype (WT) and Toso^(−/−) mice. Toso RNA wasanalyzed using RT-PCR and expressed as Expression per 18S RNA (n=4).FIG. 12B: Splenocytes from C57BL/6 mice and Toso^(−/−) mice were stainedfor CD19 (B cells) and CD3 (T cells) and co-stained with rat anti-mouseToso antibody or isotype control. Grey areas indicate isotype control,bolded lines indicate staining with anti Toso antibody. Onerepresentative histogram of CD19 positive cells (B cells) and CD3positive cells (T cells) is shown. FIG. 12C: Splenocytes and bloodleukocytes from C57BL/6 mice and Toso^(−/−) mice were stained for Gr1together with rat anti-mouse Toso antibody or isotype control. Greyareas indicate isotype control, bolded lines indicate staining withToso. One representative histogram of Gr1 positive cells is shown. FIG.12D: Blood leukocytes from wildtype and Toso^(−/−) mice were analyzedfor granulocytes and lymphocytes by forward and side scatter. Leukocytesper μl are shown (n=4). FIG. 12E: Lymphocytes were analyzed for CD4 Tcells (CD3⁺ CD8⁻ cells) CD8 T cells (CD3⁺ CD8⁺ cells) and B cells (B220⁺cells, n=4). FIG. 12F: Spleen weight from wildtype and Toso^(−/−) micewere analyzed (n=4). FIG. 12G: Spleen lymphocytes were analyzed for CD4T cells (CD3⁺ CD8⁻ cells) CD8 T cells (CD3⁺ CD8⁺ cells) and B cells(B220⁺ cells, n=4).

FIG. 13A, FIG. 13B, FIG. 13C, FIG. 13D, FIG. 13E and FIG. 13F show thatthreshold for activation is lowered in granulocytes of TOSO deficientmice. FIG. 13A: Blood from wildtype or Toso^(−/−) mice was incubatedwith different concentrations of fMLP for 30 minutes at 37° C.Activation of granulocytes was measured by ROS production(Dihydrorhodamine staining) and degranulation (side-scatter) asdescribed in methods. One representative dot plot of cells gated ongranulocytes is shown. Gate is set on activated granulocytes. Percentageof activated granulocytes is given (n=6). FIG. 13B: Blood from WT andToso^(−/−) mice was incubated with different concentrations of TNF-α.Percentage of activated granulocytes is given (n=6). FIG. 13C: Bloodfrom WT and Toso^(−/−) mice was incubated with different concentrationsof Lipopolysacharide. Percentage of activated granulocytes is given(n=6). FIG. 13D: Blood from WT and Toso^(−/−) mice was incubated withdifferent concentrations of GM-CSF (given as percent of supernatant formX63O cells). Percentage of activated granulocytes is given (n=6). FIG.13E: Blood from wildtype and Toso^(−/−) mice was primed with 10% GM-CSFsupernatant or 500 ng/ml LPS. After 30 minutes cells were stimulatedwith 2 μM fMLP for 15 minutes. Percentage of activated granulocytes isgiven (n=6). FIG. 13F: Blood from WT and Toso^(−/−) mice was incubatedat different temperatures for 30 minutes. Degranulation was measuredusing side scatter. Percentage of de-granulated cells is given (n=6).

FIG. 14A, FIG. 14B, FIG. 14C and FIG. 14D show that activation thresholdin granulocytes is regulated intrinsically. C57BL/6 wildtype mice wereirradiated and reconstituted with bone marrow form wildtype micecarrying a point mutation in the CD45 gene (CD45.1, group 1), Toso^(−/−)mice (CD45.2, group 2) or a 1:1 mixture of wildtype (CD45.1) bone marrowand Toso^(−/−) bone marrow cells (CD45.2, group 3) as describe inmethods. 30 days after bone marrow transplantation, peripheral blood wasstimulated with fMLP and analyzed for activated granulocytes FIG. 14A:Representative FACS plots of cells gated for Gr1 and stained for CD45.1,CD45.2 and Dihydrorhodamin (ROS) are shown. FIG. 14B: Percentages ofgranulocytes are quantified for the three different groups of bonemarrow chimeras (n=8). FIG. 14C: Percentages of activated granulocytesmeasured by side scatter and ROS production according to the gate inFIG. 3 a was analyzed (n=8). FIG. 14D: The mean fluorescent intensity ofside scatter and Dihydrorhodamin (ROS) was analyzed for total andactivated granulocytes (n=8).

FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D, FIG. 15E and FIG. 15F showimpaired control of Listeria in Toso deficient mice. FIG. 15A:Toso^(−/−) mice and corresponding wildtype mice were infected with 1×10⁴CFU of Listeria. Survival was monitored (n=8-15). FIG. 15B: Toso^(−/−)mice and corresponding wildtype mice were infected with 1×10⁴ CFU ofListeria. Granulocyte activation (ROS formation) was measured in bloodgranulocytes d0 and d2 after infection (n=4-5). FIG. 15C: WT andToso^(−/−) mice were infected with 1×10⁶ CFU of Listeria.Myeloperoxidase was analyzed in plasma of naïve WT mice, and Listeriainfected WT and Toso^(−/−) mice six hours after infection (n=4). FIG.15D: Toso^(−/−) mice and corresponding WT mice were infected with 1×10⁶CFU of Listeria monocytogenes. Liver histology was analyzed 20 hourslater. One representative slide is shown (n=4). Scale bar=50 μm. FIG.15E: Toso^(−/−) mice and corresponding WT mice were infected with 1×10⁴CFU of Listeria. After one day ROS production was measured ingranulocytes of spleen and liver (n=3). FIG. 15F: Toso^(−/−) mice andcorresponding WT mice were infected with 1×10⁴ CFU of Listeria. On day 3and 4 after infection, Listeria titers were analyzed in spleen, liverand brain (n=6).

FIG. 16A, FIG. 16B, FIG. 16C and FIG. 16D show that expression of CD11band CD18 is influenced by Toso. FIG. 16A: Blood granulocytes fromToso^(−/−) mice and corresponding WT mice were formalin fixed and thenstained for CD11a, CD11b and CD18. Mean fluorescent expression is shown(n=6). p values are derived from paired students t test. FIG. 16B: Bloodfrom wildtype and Toso^(−/−) mice was stimulated with differentconcentrations of GM-CSF and LPS. Mean fluorescent expression for CD11bon granulocytes is shown (n=6). FIG. 16C: Blood granulocytes fromCd11b^(−/−) mice and corresponding wildtype mice were stimulated with 2μM fMLP or with 40% GM-CSF supernatant in addition to 2 μM fMLP. Meanfluorescent intensity is given for CD11b and CD18 (n=4). FIG. 16D: BloodGranulocytes from Cd11b^(−/−) mice and corresponding wildtype mice werestimulated with 2 μM fMLP or with 40% GM-CSF supernatant in addition to2 μM fMLP. Percentage of activated granulocytes is given (n=4). Meanfluorescent intensity of ROS staining is given for total granulocytes aswell as for activated granulocytes (n=4).

FIG. 17 shows apoptosis in Toso^(−/−) granulocytes. Blood fromToso^(−/−)− was incubated at 37° C. with and without GM-CSF (10%).Apoptosis was measured with Annexin V and 7AAD after one and seven hours(n=4).

FIG. 18 shows Listerium bacteraemia after infection of Toso mice.Toso^(−/−)− and corresponding wildtype mice were infected with 1×10⁶ CFUof Listeria monocytogenes. Blood Listeria titer was assessed one and 20hours after infection (n=4).

FIG. 19A and FIG. 19B show data from wildtype and Toso^(−/−) mice afterinitiation of a high fat diet. FIG. 19A shows body weights of wildtypeand Toso^(−/−) male mice, which were monitored since the initiation of ahigh fat diet starting at 5 weeks of age. FIG. 19B shows measurements offood intake in both sets of mice at 14 weeks post high fat diet.

FIG. 20A shows data from a glucose tolerance test from wildtype andToso^(−/−) mice after initiation of a high fat diet. FIG. 20B shows datafrom an insulin tolerance test from the same set of mice.

FIG. 21A and FIG. 21B show data illustrating that wildtype andToso^(−/−) mice maintained on a regular chow (non-high fat) diet displaysimilar levels of glucose tolerance (FIG. 21A) and insulin tolerance(FIG. 21B).

FIG. 22A and FIG. 22B show data on glucose tolerance before treatment ofwildtype mice with soluble Toso protein (FIG. 22A) and after treatment(FIG. 22B).

FIG. 23 shows EAE clinical scores in mice treated with PBS (closedcircle) compared to mice treated with soluble Toso protein(Toso-Fc—closed square).

FIG. 24A shows data in an arthritis mouse model comparing the additivearthritis score in mice treated with the control vehicle (closed circle)and mouse treated with soluble Toso protein (Toso-Fc—open square). FIG.24B shows data from an arthritis mouse model comparing the percentincidence of arthritis in the Toso-Fc treated mice (Toso-Fc—opensquares) as compared to control (vehicle—closed circles).

FIG. 25 shows reduced proliferative responses to collagen in splenocytestreated with Toso-Fc.

FIG. 26A and FIG. 26B show the therapeutic effect of soluble Tosoprotein (Toso-Fc—open square) in an arthritis mouse model as compared tocontrol (vehicle—closed circle).

FIG. 27A, FIG. 27B and FIG. 27C provide sequences of differentembodiments of soluble Toso proteins of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, phage display, anddetection of hybridization using a label. Specific illustrations ofsuitable techniques can be had by reference to the example herein below.However, other equivalent conventional procedures can, of course, alsobe used. Such conventional techniques and descriptions can be found instandard laboratory manuals such as Genome Analysis: A Laboratory ManualSeries (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: ALaboratory Manual, PCR Primer: A Laboratory Manual, and MolecularCloning: A Laboratory Manual (all from Cold Spring Harbor LaboratoryPress), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York,Gait, “Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W. H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated in their entirety by reference for allpurposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a polymerase”refers to one agent or mixtures of such agents, and reference to “themethod” includes reference to equivalent steps and methods known tothose skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing devices, compositions, formulations andmethodologies which are described in the publication and which might beused in connection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the composition or method. “Consisting of” shall meanexcluding more than trace elements of other ingredients for claimedcompositions and substantial method steps. Embodiments defined by eachof these transition terms are within the scope of this invention.Accordingly, it is intended that the methods and compositions caninclude additional steps and components (comprising) or alternativelyincluding steps and compositions of no significance (consistingessentially of) or alternatively, intending only the stated method stepsor compositions (consisting of).

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1. It is to be understood, althoughnot always explicitly stated that all numerical designations arepreceded by the term “about”. The term “about” also includes the exactvalue “X” in addition to minor increments of “X” such as “X+0.1” or“X−0.1.” It also is to be understood, although not always explicitlystated, that the reagents described herein are merely exemplary and thatequivalents of such are known in the art.

A “composition” may include any substance comprising an agent orcompound and is also intended to encompass any combination of an agentor compound and other substances, including a carrier, e.g., compound orcomposition, inert (for example, a detectable agent or label) or active,such as an adjuvant, diluent, binder, stabilizer, buffers, salts,lipophilic solvents, preservative, adjuvant or the like. Carriers alsoinclude pharmaceutical excipients and additives proteins, peptides,amino acids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin such as humanserum albumin (HSA), recombinant human albumin (rHA), gelatin, casein,and the like. Representative amino acid/antibody components, which canalso function in a buffering capacity, include alanine, glycine,arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine,lysine, leucine, isoleucine, valine, methionine, phenylalanine,aspartame, and the like. Carbohydrate excipients are also intendedwithin the scope of this invention, examples of which include but arenot limited to monosaccharides such as fructose, maltose, galactose,glucose, D-mannose, sorbose, and the like; disaccharides, such aslactose, sucrose, trehalose, cellobiose, and the like; polysaccharides,such as raffinose, melezitose, maltodextrins, dextrans, starches, andthe like; and alditols, such as mannitol, xylitol, maltitol, lactitol,xylitol sorbitol (glucitol) and myoinositol.

The term pharmaceutically acceptable carrier (or medium), which may beused interchangeably with the term biologically compatible carrier ormedium, refers to reagents, cells, compounds, materials, compositions,and/or dosage forms that are not only compatible with the cells andother agents to be administered therapeutically, but also are, withinthe scope of sound medical judgment, suitable for use in contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other complication commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable carrierssuitable for use in the present invention include liquids, semi-solid(e.g., gels) and solid materials (e.g., cell scaffolds and matrices,tubes sheets and other such materials as known in the art and describedin greater detail herein). These semi-solid and solid materials may bedesigned to resist degradation within the body (non-biodegradable) orthey may be designed to degrade within the body (biodegradable,bioerodable). A biodegradable material may further be bioresorbable orbioabsorbable, i.e., it may be dissolved and absorbed into bodily fluids(water-soluble implants are one example), or degraded and ultimatelyeliminated from the body, either by conversion into other materials orbreakdown and elimination through natural pathways.

As used herein, the term “patient” or “subject” intends an animal, amammal or yet further a human patient. For the purpose of illustrationonly, a mammal includes but is not limited to a human, a simian, amurine, a bovine, an equine, a porcine or an ovine.

As used herein, the term “oligonucleotide” or “polynucleotide” refers toa short polymer composed of deoxyribonucleotides, ribonucleotides or anycombination thereof. Oligonucleotides are generally at least about 10,15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides inlength. An oligonucleotide may be used as a primer or as a probe.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that function in amanner similar to a naturally occurring amino acid.

The term “isolated” as used herein refers to molecules or biological orcellular materials being substantially free from other materials, e.g.,greater than 70%, or 80%, or 85%, or 90%, or 95%, or 98%. In one aspect,the term “isolated” refers to nucleic acid, such as DNA or RNA, orprotein or polypeptide, or cell or cellular organelle, or tissue ororgan, separated from other DNAs or RNAs, or proteins or polypeptides,or cells or cellular organelles, or tissues or organs, respectively,that are present in the natural source and which allow the manipulationof the material to achieve results not achievable where present in itsnative or natural state, e.g., recombinant replication or manipulationby mutation. The term “isolated” also refers to a nucleic acid orpeptide that is substantially free of cellular material, viral material,or culture medium when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized.Moreover, an “isolated nucleic acid” is meant to include nucleic acidfragments which are not naturally occurring as fragments and would notbe found in the natural state. The term “isolated” is also used hereinto refer to polypeptides which are isolated from other cellular proteinsand is meant to encompass both purified and recombinant polypeptides,e.g., with a purity greater than 70%, or 80%, or 85%, or 90%, or 95%,98%, or 99%. The term “isolated” is also used herein to refer to cellsor tissues that are isolated from other cells or tissues and is meant toencompass both cultured and engineered cells or tissues.

A “recombinant” nucleic acid refers an artificial nucleic acid that iscreated by combining two or more sequences that would not normally occurtogether. In one embodiment, it is created through the introduction ofrelevant DNA into an existing organismal DNA, such as the plasmids ofbacteria, to code for or alter different traits for a specific purpose,such as antibiotic resistance. A “recombinant” polypeptide is apolypeptide that is derived from a recombinant nucleic acid.

As used herein, the term “promoter” refers to a nucleic acid sequencesufficient to direct transcription of a gene. Also included in theinvention are those promoter elements which are sufficient to renderpromoter dependent gene expression controllable for cell type specific,tissue specific or inducible by external signals or agents.

In some embodiments, a promoter is an inducible promoter or a discretepromoter. Inducible promoters can be turned on by a chemical or aphysical condition such as temperature or light. Examples of chemicalpromoters include, without limitation, alcohol-regulated,tetracycline-regulated, steroid-regulated, metal-regulated andpathogenesis-related promoters. Examples of discrete promoters can befound in, for examples, Wolfe et al. Molecular Endocrinology 16(3):435-49.

As used herein, the term “regulatory element” refers to a nucleic acidsequence capable of modulating the transcription of a gene. Non-limitingexamples of regulatory element include promoter, enhancer, silencer,poly-adenylation signal, transcription termination sequence. Regulatoryelement may be present 5′ or 3′ regions of the native gene, or within anintron.

Various proteins are also disclosed herein with their GenBank AccessionNumbers for their human proteins and coding sequences. However, theproteins are not limited to human-derived proteins having the amino acidsequences represented by the disclosed GenBank Accession Nos, but mayhave an amino acid sequence derived from other animals, particularly, awarm-blooded animal (e.g., rat, guinea pig, mouse, chicken, rabbit, pig,sheep, cow, monkey, etc.).

As used herein, the term “Toso”, “FAIM3” or “Fas apoptotic inhibitorymolecule 3” refers to a protein having an amino acid sequencesubstantially identical to any of the representative Toso sequences,including any and all versions of GenBank Accession Nos. NP_(—)001135945(human isoform b), NP_(—)001180267 (human isoform c), NP_(—)005440(human isoform a), NP_(—)081252 (mouse) or NP_(—)001014843 (rat).Suitable cDNA encoding Toso are provided at GenBank Accession Nos.NM_(—)001142473, NM_(—)001193338, NM_(—)005449, NM_(—)026976, andNM_(—)001014843.

As used herein, the term “biological activity of Toso” or “Tosoactivity” refers to any biological activity associated with the fulllength native Toso protein. In some embodiments, the biological activityof Toso refers to binding to an IgM antibody. In further embodiments,the biological activity of Toso refers to inhibiting CD11b or CD18activity. In yet further embodiments, the biological activity of Tosorefers to increasing the activation threshold of granulocytes.Activation threshold can be measured by number of activated granulocytesfrom bone marrow. In further embodiments, the biological activity ofToso includes the activation of dendritic cells and their ability topresent antigen to T cells. In further embodiments, the biologicalactivity of Toso includes inhibition of apoptosis or enhancement of TNFsignaling. In some embodiments, the Toso biological activity isequivalent to the activity of a protein having an amino acid sequencerepresented by GenBank Accession No. NP_(—)001135945, NP_(—)001180267,NP_(—)005440, NP_(—)081252 or NP_(—)001014843, including any and allversions of these accession numbers.

As used herein, the term “CD11b”, “ITGAM” or “ITGAM integrin, alpha M(complement component 3 receptor 3 subunit)” refers to a protein havingan amino acid sequence substantially identical to the representativeCD11b sequence of GenBank Accession No. NP_(—)000623. A suitable cDNAencoding CD11b is provided at GenBank Accession No. NM_(—)000632.

As used herein, the term “biological activity of CD11b” refers to anybiological activity associated with the full length native CD11bprotein. In one embodiment, the biological activity of CD11b refers tocombining with the beta 2 chain (ITGB2) to form a leukocyte-specificintegrin. In suitable embodiments, the CD11 b biological activity isequivalent to the activity of a protein having an amino acid sequencerepresented by GenBank Accession No. NP_(—)000623. Measurement oftranscriptional activity can be performed using any known method, suchas immunohistochemistry, reporter assay or RT-PCR.

As used herein, the term “CD18”, “ITGB2” or “ITGB2 integrin, beta 2(complement component 3 receptor 3 and 4 subunit)” refers to a proteinhaving an amino acid sequence substantially identical to therepresentative CD18 sequence of GenBank Accession No. NP_(—)000202. Asuitable cDNA encoding CD18 is provided at GenBank Accession No.NM_(—)000211.

As used herein, the term “treating” refers to administering apharmaceutical composition for the purpose of improving the condition ofa patient by reducing, alleviating, reversing, or preventing at leastone adverse effect or symptom of a disease or disorder.

As used herein, the term “preventing” refers to identifying a subject(i.e., a patient) having an increased susceptibility to a disease butnot yet exhibiting symptoms of the disease, and administering a therapyaccording to the principles of this disclosure. The preventive therapyis designed to reduce the likelihood that the susceptible subject willlater become symptomatic or that the disease will be delay in onset orprogress more slowly than it would in the absence of the preventivetherapy. A subject may be identified as having an increased likelihoodof developing the disease by any appropriate method including, forexample, by identifying a family history of the disease or otherdegenerative brain disorder, or having one or more diagnostic markersindicative of disease or susceptibility to disease.

As used herein, the term “sample” or “test sample” refers to any liquidor solid material containing nucleic acids. In suitable embodiments, atest sample is obtained from a biological source (i.e., a “biologicalsample”), such as cells in culture or a tissue sample from an animal,most preferably, a human.

As used herein, the term “substantially identical”, when referring to aprotein or polypeptide, is meant one that has at least 80%, 85%, 90%,95%, or 99% sequence identity to a reference amino acid sequence. Thelength of comparison is preferably the full length of the polypeptide orprotein, but is generally at least 10, 15, 20, 25, 30, 40, 50, 60, 80,or 100 or more contiguous amino acids. A “substantially identical”nucleic acid is one that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% sequence identity to a reference nucleic acid sequence. Thelength of comparison is preferably the full length of the nucleic acid,but is generally at least 20 nucleotides, 30 nucleotides, 40nucleotides, 50 nucleotides, 75 nucleotides, 100 nucleotides, 125nucleotides, or more.

As used herein, an “amino acid substitution” or “substitution” refers tothe replacement of an amino acid at a particular position in a startingpolypeptide sequence with another amino acid. For example, thesubstitution M23Y refers to a variant polypeptide in which themethionine at position 23 is replaced with a tyrosine.

A “biological equivalent” of a protein or nucleic acid refers to aprotein or nucleic acid that is substantially identical to the proteinor nucleic acid by amino acid or nucleic acid sequence or that has anequivalent biological activity.

As used herein, the term “effective amount” refers to a quantity ofcompound (e.g., a Toso protein or biologically active fragment thereof)delivered with sufficient frequency to provide a medical benefit to thepatient. In one embodiment, an effective amount of a protein is anamount sufficient to treat or ameliorate a symptom of a disease.

A population of cells intends a collection of more than one cell that isidentical (clonal) or non-identical in phenotype and/or genotype.

“Substantially homogeneous” describes a population of cells in whichmore than about 50%, or alternatively more than about 60%, oralternatively more than 70%, or alternatively more than 75%, oralternatively more than 80%, or alternatively more than 85%, oralternatively more than 90%, or alternatively, more than 95%, of thecells are of the same or similar phenotype. Phenotype can be determinedby a pre-selected cell surface marker or other marker.

The terms autologous transfer, autologous transplantation, autograft andthe like refer to treatments wherein the cell donor is also therecipient of the cell replacement therapy. The terms allogeneictransfer, allogeneic transplantation, allograft and the like refer totreatments wherein the cell donor is of the same species as therecipient of the cell replacement therapy, but is not the sameindividual. A cell transfer in which the donor's cells and have beenhistocompatibly matched with a recipient is sometimes referred to as asyngeneic transfer. The terms xenogeneic transfer, xenogeneictransplantation, xenograft and the like refer to treatments wherein thecell donor is of a different species than the recipient of the cellreplacement therapy.

As used herein, an “antibody” includes whole antibodies and any antigenbinding fragment or a single chain thereof. Thus the term “antibody”includes any protein or peptide containing molecule that comprises atleast a portion of an immunoglobulin molecule. Examples of such include,but are not limited to a complementarity determining region (CDR) of aheavy or light chain or a ligand binding portion thereof, a heavy chainor light chain variable region, a heavy chain or light chain constantregion, a framework (FR) region, or any portion thereof, or at least oneportion of a binding protein. In general, the term “antibody” includesany polypeptide that includes at least one constant domain, including,but not limited to, CH1, CH2, CH3 and CL. Antibodies that find use inthe present invention can take on a number of formats as describedherein, including traditional antibodies as well as antibodyderivatives, fragments and mimetics.

The antibodies can be polyclonal or monoclonal and can be isolated fromany suitable biological source, e.g., murine, rat, sheep and canine.

A monoclonal antibody is an antibody produced by a single clone of cellsor a hybridoma, and therefore is a single pure homogeneous type ofantibody.

A hybridoma is a cell that is produced in the laboratory from the fusionof an antibody-producing lymphocyte and a non-antibody producing cancercell, usually a myeloma or lymphoma. A hybridoma proliferates andproduces a continuous supply of a specific monoclonal antibody.

The term “human antibody” as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody” as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. Thus, as used herein, the term “human antibody”refers to an antibody in which substantially every part of the protein(e.g., CDR, framework, C_(L), C_(H) domains (e.g., C_(H1), C_(H2),C_(H3)), hinge, (VL, VH)) is substantially non-immunogenic in humans,with only minor sequence changes or variations. Similarly, antibodiesdesignated primate (monkey, baboon, chimpanzee, etc.), rodent (mouse,rat, rabbit, guinea pig, hamster, and the like) and other mammalsdesignate such species, sub-genus, genus, sub-family, family specificantibodies. Further, chimeric antibodies include any combination of theabove. Such changes or variations optionally and preferably retain orreduce the immunogenicity in humans or other species relative tonon-modified antibodies. Thus, a human antibody is distinct from achimeric or humanized antibody. It is pointed out that a human antibodycan be produced by a non-human animal or prokaryotic or eukaryotic cellthat is capable of expressing functionally rearranged humanimmunoglobulin (e.g., heavy chain and/or light chain) genes. Further,when a human antibody is a single chain antibody, it can comprise alinker peptide that is not found in native human antibodies. Forexample, an Fv can comprise a linker peptide, such as two to about eightglycine or other amino acid residues, which connects the variable regionof the heavy chain and the variable region of the light chain. Suchlinker peptides are considered to be of human origin.

As used herein, a human antibody is “derived from” a particular germlinesequence if the antibody is obtained from a system using humanimmunoglobulin sequences, e.g., by immunizing a transgenic mousecarrying human immunoglobulin genes or by screening a humanimmunoglobulin gene library. A human antibody that is “derived from” ahuman germline immunoglobulin sequence can be identified as such bycomparing the amino acid sequence of the human antibody to the aminoacid sequence of human germline immunoglobulins. A selected humanantibody typically is at least 90% identical in amino acids sequence toan amino acid sequence encoded by a human germline immunoglobulin geneand contains amino acid residues that identify the human antibody asbeing human when compared to the germline immunoglobulin amino acidsequences of other species (e.g., murine germline sequences). In certaincases, a human antibody may be at least 95%, or even at least 96%, 97%,98%, or 99% identical in amino acid sequence to the amino acid sequenceencoded by the germline immunoglobulin gene. Typically, a human antibodyderived from a particular human germline sequence will display no morethan 10 amino acid differences from the amino acid sequence encoded bythe human germline immunoglobulin gene. In certain cases, the humanantibody may display no more than 5, or even no more than 4, 3, 2, or 1amino acid difference from the amino acid sequence encoded by thegermline immunoglobulin gene.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the antibody, e.g., from a transfectoma,antibodies isolated from a recombinant, combinatorial human antibodylibrary, and antibodies prepared, expressed, created or isolated by anyother means that involve splicing of human immunoglobulin gene sequencesto other DNA sequences. Such recombinant human antibodies have variableand constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo. Methods to makingthese antibodies are described herein.

“Isotype” as used herein is meant any of the subclasses ofimmunoglobulins defined by the chemical and antigenic characteristics oftheir constant regions. It should be understood that therapeuticantibodies can also comprise hybrids of isotypes and/or subclasses.

The terms “polyclonal antibody” or “polyclonal antibody composition” asused herein refer to a preparation of antibodies that are derived fromdifferent B-cell lines. They are a mixture of immunoglobulin moleculessecreted against a specific antigen, each recognizing a differentepitope.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

As used herein, the term “label” intends a directly or indirectlydetectable compound or composition that is conjugated directly orindirectly to the composition to be detected, e.g., N-terminal histidinetags (N-His), magnetically active isotopes, e.g., ¹¹⁵Sn, ¹¹⁷Sn and¹¹⁹Sn, a non-radioactive isotopes such as ¹³C and ¹⁵N, polynucleotide orprotein such as an antibody so as to generate a “labeled” composition.The term also includes sequences conjugated to the polynucleotide thatwill provide a signal upon expression of the inserted sequences, such asgreen fluorescent protein (GFP) and the like. The label may bedetectable by itself (e.g. radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition which is detectable. The labelscan be suitable for small scale detection or more suitable forhigh-throughput screening. As such, suitable labels include, but are notlimited to magnetically active isotopes, non-radioactive isotopes,radioisotopes, fluorochromes, chemiluminescent compounds, dyes, andproteins, including enzymes. The label may be simply detected or it maybe quantified. A response that is simply detected generally comprises aresponse whose existence merely is confirmed, whereas a response that isquantified generally comprises a response having a quantifiable (e.g.,numerically reportable) value such as an intensity, polarization, and/orother property. In luminescence or fluorescence assays, the detectableresponse may be generated directly using a luminophore or fluorophoreassociated with an assay component actually involved in binding, orindirectly using a luminophore or fluorophore associated with another(e.g., reporter or indicator) component.

Examples of luminescent labels that produce signals include, but are notlimited to bioluminescence and chemiluminescence. Detectableluminescence response generally comprises a change in, or an occurrenceof, a luminescence signal. Suitable methods and luminophores forluminescently labeling assay components are known in the art anddescribed for example in Haugland, Richard P. (1996) Handbook ofFluorescent Probes and Research Chemicals (6^(th) ed.). Examples ofluminescent probes include, but are not limited to, aequorin andluciferases.

Examples of suitable fluorescent labels include, but are not limited to,fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, LuciferYellow, Cascade Blue™, and Texas Red. Other suitable optical dyes aredescribed in the Haugland, Richard P. (1996) Handbook of FluorescentProbes and Research Chemicals (6^(th) ed.).

In another aspect, the fluorescent label is functionalized to facilitatecovalent attachment to a cellular component present in or on the surfaceof the cell or tissue such as a cell surface marker. Suitable functionalgroups, including, but not are limited to, isothiocyanate groups, aminogroups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonylhalides, all of which may be used to attach the fluorescent label to asecond molecule. The choice of the functional group of the fluorescentlabel will depend on the site of attachment to either a linker, theagent, the marker, or the second labeling agent.

Although the present invention is described primarily with reference tospecific embodiments, it is also envisioned that other embodiments willbecome apparent to those skilled in the art upon reading the presentdisclosure, and it is intended that such embodiments be contained withinthe present inventive methods.

I. Overview of the invention

The present invention is directed to methods and compositions formodulating the activity of the Toso protein (which is alsointerchangeably referred to herein as “Toso” or “Toso receptor” or“Faim3” or “FCMR”). In some embodiments, the methods and compositions ofthe invention increase activity of the Toso protein. In otherembodiments, the methods and compositions of the invention inhibitactivity of the Toso protein. In some embodiments, compositions formodulating the activity of the Toso protein include agents that bind tothe Toso protein or to a ligand of the Toso protein. In furtherembodiments, the compositions of the invention include a soluble Tosoprotein. As will be discussed in further detail herein, soluble Tosoproteins of the invention include all or part of the extracellulardomain of a Toso receptor. Soluble Toso proteins of the invention mayfurther include a signal peptide and/or an Fc domain. Soluble Tosoproteins of the invention may further include variant extracellulardomains of a Toso protein, including deletion variants (variants inwhich one or more amino acids of the full extracellular domain aredeleted) and variants comprising one or more amino acid substitutions.

The present invention is further directed to methods of treatingdisorders and diseases by administering a soluble Toso protein (or avariant thereof) to a subject. As will be discussed in further detailherein, soluble Toso proteins of the invention can be used to treatsubjects suffering from without limitation an autoimmune disorder(including without limitation Type 1 or Type 2 diabetes, multiplesclerosis, or rheumatoid arthritis), asthma, allergy, chronicobstructive pulmonary disease (“COPD”), hyper-IgM syndrome, CLL, lupus,or a neutrophilia-associated disorder (including without limitationneutropenia, severe congenital neutropenia, cyclical neutropenia,antibody mediated neutropenia, reticular dysgenesis, leukocyte adhesiondeficiency, familiar myeloproliferative disease, chronic myelogenousleukemia, familiar cold urticaria and leukocytosis, and chronicgranulomatous disease).

II. Soluble Toso Protein

Compositions of the invention include agents that modulate Tosoactivity. Such compositions, as will be discussed in further detailbelow, include without limitation a soluble form of the Toso protein.

A soluble Toso protein of the invention (also referred tointerchangeably herein as the “soluble Toso receptor,” “Toso-Fc”, and“soluble Toso polypeptide”) includes all or part of an extracellulardomain of a Toso receptor. The soluble Toso proteins of the invention infurther embodiments include a signal domain and/or an Fc domain. As willbe discussed in further detail herein, these components of the solubleToso protein may be combined in any way with or without additionalcomponents and/or modifications to provide a soluble Toso protein of theinvention.

In one aspect, the soluble Toso protein of the invention comprises anextracellular domain of Toso. In a still further embodiments, thesoluble Toso protein comprises the extracellular domain of human Tosoisoform a. The extracellular domain of human Toso is predicted to spanamino acids P21 to G251 of NP_(—)005440.1, human Toso isoform a (seeShima et al., Int. Immunol., 2010, which is hereby incorporated in itsentirety for all purposes and in particular for all teachings related tothe extracellular domain of Toso). For the sake of clarity, the majorityof the discussion herein is directed to soluble Toso proteins comprisingall or part of an extracellular domain of a human Toso protein. However,it will be appreciated that the extracellular domain of a Toso proteinfrom any species can be used to produce soluble Toso proteins inaccordance with the description herein, and it would be well within theability of one of skill in the art to identify the regions of the Tosoprotein from another species that correspond to the regions of the humanToso protein discussed herein.

In one embodiment, the soluble Toso protein comprises an extracellulardomain sequence according to SEQ ID NO: 1, which is shown in FIG. 10. Ina further embodiment, the soluble Toso protein has a sequence identityof about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% and 100% identity to SEQ ID NO: 1. In a still furtherembodiment, the soluble Toso protein comprises a polypeptide with 1-75,2-70, 3-65, 4-60, 5-55, 6-50, 7-45, 8-40, 9-35, 10-30, 11-25, 12-20,13-15, 5-20, 6-18, 8-16, 10-14 amino acid substitutions in SEQ ID NO: 1.In a yet further embodiments, the soluble Toso protein comprises apolypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acidsubstitutions in SEQ ID NO: 1.

In one embodiment, the soluble Toso protein comprises an extracellulardomain sequence according to SEQ ID NO: 8, which is shown in FIG. 27. Ina further embodiment, the soluble Toso protein has a sequence identityof about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% and 100% identity to SEQ ID NO: 8. In a still furtherembodiment, the soluble Toso protein comprises a polypeptide with 1-75,2-70, 3-65, 4-60, 5-55, 6-50, 7-45, 8-40, 9-35, 10-30, 11-25, 12-20,13-15, 5-20, 6-18, 8-16, 10-14 amino acid substitutions in SEQ ID NO: 8.In a yet further embodiments, the soluble Toso protein comprises apolypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acidsubstitutions in SEQ ID NO: 8.

In a further embodiment and in accordance with any of the above, thesoluble Toso protein of the invention includes amino acids 18 to 253 ofSEQ ID NO: 7. In a still further embodiment, the soluble Toso protein ofthe invention includes amino acids 21 to 253 of SEQ ID NO: 7. In a stillfurther embodiment, the soluble Toso protein includes amino acids 21 to251 of SEQ ID NO: 7. In a yet further embodiment, the soluble Tosoprotein includes any of the following ranges of amino acids from SEQ IDNO: 7: 1-255, 5-245, 10-235, 15-225, 20-215, 25-205, 30-195, 35-185,40-175, 45-165, 50-155, 45-145, 40-135, 35-125, 30-115, 35-105, 40-95,45-85, 50-75, 55-65. In a still further embodiment, the soluble Tosoprotein includes a sequence with a sequence identity of about 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%identity to amino acids 18 to 253 or 21 to 253 of SEQ ID NO: 7.

In a still further embodiment and in accordance with any of the above,the soluble Toso protein of the invention includes all or a portion ofSEQ ID NO: 8, pictured in FIG. 27. In still further embodiments, thesoluble Toso protein includes amino acids 1-231, 6-221, 11-211, 16-201,21-191, 26-181, 31-171, 36-161, 41-151, 46-141, 51-131, 56-121, 61-111,66-101, 71-91, 76-81 of SEQ ID NO: 8. In yet further embodiments, thesoluble Toso protein includes a polypeptide with at least 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% sequenceidentity to the amino acid regions 1-231, 6-221, 11-211, 16-201, 21-191,26-181, 31-171, 36-161, 41-151, 46-141, 51-131, 56-121, 61-111, 66-101,71-91, 76-81 of SEQ ID NO: 8.

In a yet further embodiment, the soluble Toso protein of the inventioncomprises any one of SEQ ID NOs: 8, 9, 11, 13, 15, 17, 19, 21, and 23.In a still further embodiment, the soluble Toso protein includes asequence with a sequence identity of about 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identity to SEQ ID NOs:8, 9, 11, 13, 15, 17, 19, 21, and 23. These sequences include deletionvariants of the extracellular domain of the Toso receptor.

In still further embodiments and in accordance with any of the above,the soluble Toso protein of the invention comprises a deletion variantof the full extracellular domain of the Toso protein. In exemplaryembodiments, the deletion variants that are a component of a solubleToso protein of the invention include a polypeptide in which one or moreof the following amino acids have been deleted from SEQ ID NO:8: 1-21,1-35, 1-87, both regions 1-21 and 211-231, 211-231, 154-231, 105-231,and 93-231. In further exemplary embodiments, the deletion variants thatare a component of a soluble Toso protein of the invention include apolypeptide with a sequence identity of about 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to a polypeptide accordingto SEQ ID NO:8 with one or more of the following amino acid regionsdeleted: 1-21, 1-35, 1-87, both regions 1-21 and 211-231, 211-231,154-231, 105-231, and 93-231.

In further embodiments and in accordance with any of the above, thesoluble Toso protein of the invention includes an extracellular domaincomponent that comprises regions that bind to a ligand of the Tosoreceptor. In an exemplary embodiment, the soluble Toso protein of theinvention includes an extracellular domain component that binds to IgM.In a still further embodiment, the soluble Toso protein of the inventionincludes amino acids 35 to 87 of SEQ ID NO: 8. In further exemplaryembodiments, the soluble Toso protein of the invention includes aminoacids 25-100, 29-95, 33-90, 37-85, 41-80, 45-75, 49-70, 53-65, or 57-60of SEQ ID NO: 8.

In a further aspect and in accordance with any of the above, the solubleToso protein of the invention includes an extracellular domain componentas is discussed above and further includes a signal sequence. In anexemplary embodiment, the signal sequence enhances secretion from hostcells. In a yet further embodiment, the signal sequence includes withoutlimitation a member selected from an IL-2 signal sequence, α-matingfactor pre-sequence from Saccharomyces cerevisiae, α-amylase signalsequence from Aspergillus niger, Glucoamylase signal sequence fromAspergillus awamori, Serum albumin signal sequence from Homo sapiens,Inulinase signal sequence from Kluyveromcyes maxianus, Invertase signalsequence from Saccharomyces cerevisiae, Killer protein signal sequencefrom Saccharomyces cerevisiae, Lysozyme signal sequence from Gallusgallus. In a still further embodiment, the signal sequence comprises asequence according to SEQ ID NO: 2, which is shown in FIG. 10. In astill further embodiment, the signal sequence comprises a sequence witha sequence identity of about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% and 100% identity to SEQ ID NO: 2. Inspecific embodiments, the soluble Toso protein of the inventioncomprises any one of SEQ ID NOs: 1, 8, 9, 11, 13, 15, 17, 21 and 23 orvariants of those sequences as a fusion protein with SEQ ID NO: 2 orwith a sequence with an identity of about 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identity to SEQ ID NO:2.

In a further aspect and in accordance with any of the above, a solubleToso protein of the invention comprises an extracellular domain of aToso receptor and an Fc domain. In an exemplary embodiment, the solubleToso protein is a fusion protein comprising the extracellular domain ofisoform A of the human Toso protein and an Fc domain. In a furtherembodiment, the Fc domain includes any domain that enhances thehalf-life of the protein as compared to the protein without the Fcdomain. In a still further embodiment, the Fc domain includes any domainthat improves the pharmacokinetic profile of the protein as compared tothe protein without the Fc domain. In a still further embodiment, the Fcdomain is derived from human IgG1 at the C-terminus, which in a yetfurther embodiment includes mutations that diminish or ablateantibody-dependent and complement dependent cytotoxicity. In a stillfurther embodiment, such mutations include one or more of the followingmutations singly or in any combination: E233P; L234V; L235A; ΔG236;A327G; A330S; P331S. In a yet further embodiment, the Fc domaincomprises a sequence according to SEQ ID NO: 3, which is shown in FIG.10. In a still further embodiment, the Fc domain comprises a sequencewith a sequence identity of about 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identity to SEQ ID NO: 3.

In further exemplary embodiments, the soluble Toso protein of theinvention comprises a fusion protein comprising both an extracellulardomain component and an Fc domain component. In still further exemplaryembodiments, the soluble Toso protein of the invention comprises SEQ IDNOs: 1, 8, 9, 11, 13, 15, 17, 21 and 23 or variants of those sequencesas a fusion protein with SEQ ID NO: 3 or with a sequence with anidentity of about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% and 100% identity to SEQ ID NO: 3.

In one aspect and in accordance with any of the above, the soluble Tosoprotein of the invention comprises an extracellular Toso domain, asignal sequence and an Fc domain—each of those components may compriseany of the above described versions of these components in anycombination. In a still further embodiment, the soluble Toso protein ofthe invention comprises a sequence according to SEQ ID NO: 5, which isshown in FIG. 8. In a still further embodiment, the soluble Toso proteinof the invention comprises a sequence with a sequence identity of about70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and100% identity to SEQ ID NO: 5.

In further embodiments and in accordance with any of the above, thesoluble Toso protein of the invention comprises a sequence according toany one of SEQ ID NOs. 10, 12, 14, 16, 18, 20, 22 and 24, which comprisean extracellular domain component, a signal sequence, and an Fc domain.In further embodiments, the soluble Toso protein of the inventioncomprises a sequence with a sequence identity of about 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% to anyone of SEQ ID NOs. 10, 12, 14, 16, 18, 20, 22 and 24.

In further aspects and in accordance with any of the above, a solubleToso protein of the invention may further include a linker between theToso extracellular domain component and the Fc domain, between the Tosoextracellular domain component and the signal sequence, or between boththe Toso extracellular domain component and the Fc domain and the Tosoextracellular domain component and the signal sequence. In exemplaryembodiments, such a linker may be an amino acid linker, a polymericlinker, or any other linker known in the art to be effective for joiningtwo amino acid sequences together. In further exemplary embodiments, thelinker is an amino acid linker. In still further embodiments, the linkeris the amino acid sequence: ISAMVRS (SEQ ID NO: 25). In yet furtherembodiments, the linker is a variant of SEQ ID NO: 25 containing 1, 2,3, 4, 5, or 6 amino acid substitutions.

In further embodiments and in accordance with any of the above, variantsof the soluble Toso proteins of the invention can be made throughmodification of the amino acid sequences of any of the soluble Tosoproteins discussed herein, including SEQ ID NOs. 5, 6, or 8-24. Suchmodifications can be achieved using any known technique in the art e.g.,site-directed mutagenesis or PCR based mutagenesis. Such techniques aredescribed for example in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989 andAusubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, New York, N.Y., 1989, each of which is incorporated by referencein its entirety for all purposes and in particular for all teachingsrelated to forming protein variants.

In still further embodiments and in accordance with any of the above,soluble Toso proteins of the invention may be in monomeric or multimericforms, wherein each monomer of the multimer comprises a singleextracellular domain sequence. In further embodiments, the soluble Tosoproteins of the invention are in multimers of 2, 3, 4, 5, 6, 7, 8, 9, 10or more monomers. In a specific embodiment, soluble Toso proteins aremultimers of 6 monomers. In a further embodiment, a hexameric Tosomultimer is formed of monomers comprising a hexamerization tag. In afurther embodiment, the hexamerization tag comprises a 21 amino acidtail piece from human IgA alpha heavy chain constant region as describedin Hirano et al., Blood (2006), which is hereby incorporated byreference for all purposes and in particular for all teachings relatedto hexamerization or other multimerization tags. In yet a furtherembodiment, the hexamerization tag of the invention includes a sequenceaccording to SEQ ID NO: 4, shown in FIG. 10.

In further embodiments and in accordance with any of the above, thesoluble Toso protein of the invention is modified to alter one or morefunctional properties of the protein. In exemplary embodiments, thesoluble Toso protein is chemically modified. For example, the solubleToso protein may be modified with one or more polymers to improve itsstability in vivo and/or alter its pharmacokinetic profile. Suchpolymers include without limitation one or more of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337,which are all hereby incorporated by reference in their entirety for allpurposes and in particular for all teachings related to linking proteinsto polymers.

Modifications of the Toso soluble protein included within the scope ofthis invention include reacting targeted amino acid residues of a Tosopolypeptide in accordance with any of the sequences and soluble Tosoproteins discussed above with an organic derivatizing agent that iscapable of reacting with selected side chains or the N- or C-terminalresidues of a Toso polypeptide. Commonly used crosslinking agentsinclude, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutarnyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of the“-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins; Structure and Molecular Properties, W. H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of modification of the soluble Toso protein included withinthe scope of this invention comprises altering the native glycosylationpattern of the polypeptide. “Altering the native glycosylation pattern”is intended for purposes herein to mean deleting one or morecarbohydrate moieties found in native sequence Toso polypeptide, and/oradding one or more glycosylation sites that are not present in thenative sequence Toso polypeptide.

Addition of glycosylation sites to Toso soluble protein may beaccomplished by altering the amino acid sequence thereof. The alterationmay be made, for example, by the addition of, or substitution by, one ormore serine or threonine residues to the native sequence the solubleToso protein (for O-linked glycosylation sites). The soluble Tosoprotein amino acid sequence may optionally be altered through changes atthe DNA level, particularly by mutating the DNA encoding the Tososoluble protein at preselected bases such that codons are generated thatwill translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theToso soluble protein is by chemical or enzymatic coupling of glycosidesto the polypeptide. Such methods are described in the art, e.g., in WO87/05330 published Sep. 11, 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981), which are hereby incorporated byreference in their entirety for all purposes and in particular for allteachings related to altering carbohydrate moieties on a protein.

Removal of carbohydrate moieties present on the soluble Toso protein maybe accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge, et al., Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura, et al., Meth. Enzymol.,138:350 (1987), which are hereby incorporated by reference in theirentirety for all purposes and in particular for all teachings related toaltering carbohydrate moieties on a protein.

The soluble Toso protein of the present invention may also be modifiedin a way to form chimeric molecules comprising the protein fused toanother, heterologous polypeptide or amino acid sequence. In oneembodiment, such a chimeric molecule comprises a fusion of a solubleToso polypeptide with a tag polypeptide which provides an epitope towhich an anti-tag antibody can selectively bind. The epitope tag isgenerally placed at the amino- or carboxyl-terminus of the Tosopolypeptide. The presence of such epitope-tagged forms of a Tosopolypeptide can be detected using an antibody against the tagpolypeptide. Also, provision of the epitope tag enables the Tosopolypeptide to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. In an alternative embodiment, the chimeric molecule maycomprise a fusion of a Toso polypeptide with an immunoglobulin or aparticular region of an immunoglobulin. For a bivalent form of thechimeric molecule, such a fusion could be to the Fc region of an IgGmolecule or GST fusions.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field, et al., Mol. Cell Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7, and 9E10antibodies thereto [Evan, et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky, et al., Protein Engineering,3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide[Hopp, et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitopepeptide [Martin, et al., Science, 255:192-194 (1992)]; tubulin epitopepeptide [Skinner, et al., J. Biol. Chem., 266:15163-15166 (1991)]; andthe T7 gene 10 protein peptide tag [Lutz-Freyermuth, et al., Proc. Natl.Acad. Sci. USA, 87; 6393-6397 (1990)].

In further embodiments and in accordance with any of the above, asoluble Toso protein of the invention is fused with a cell penetratingpeptide.

In further aspects, the present invention encompasses nucleic acidsencoding one or more soluble Toso proteins as well as host cellscomprising such nucleic acids. In certain embodiments, host cells usedin accordance with the present invention include without limitationHEK293F, HEK298T, Cos7, HeLa, and CHO-DHFR deficient cells. In stillfurther embodiments, soluble Toso proteins of the invention are stablyexpressed in a cell line that has been modified to grow in a serum-freesuspension.

In further aspects, compositions of the invention may include any of thesoluble Toso proteins discussed herein along with additives andpharmaceutically acceptable carriers. As used herein, “pharmaceuticallyacceptable carrier” includes any material, which when combined with theconjugate retains the conjugates' activity and is non-reactive with thesubject's immune systems. Examples include, but are not limited to, anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Other carriers may also include sterilesolutions, tablets including coated tablets and capsules. Typically suchcarriers contain excipients such as starch, milk, sugar, certain typesof clay, gelatin, stearic acid or salts thereof, magnesium or calciumstearate, talc, vegetable fats or oils, gums, glycols, or other knownexcipients. Such carriers may also include flavor and color additives orother ingredients. Compositions comprising such carriers are formulatedby well known conventional methods.

III. Antibodies to Toso

In one aspect, the present invention provides an antibody that binds tothe Toso protein. In some embodiments, antibodies of the inventionincrease Toso activity. In other embodiments, antibodies of theinvention decrease Toso activity.

Methods of preparing antibodies are generally known in the art. Forexample, U.S. Pat. No. 6,727,350 discloses an antibody directed to Toso,which is hereby incorporated by reference in its entirety for allpurposes and in particular for all teachings related to antibodiesdirected to the Toso protein.

An antibody of the invention may be a polyclonal antibody, monoclonalantibody, chimeric antibody, humanized antibody or a derivative orfragment thereof as defined below. In one aspect, a fragment comprises,or alternatively consists essentially of, or yet further consists of theCDR of an antibody. In one aspect, an antibody of the invention isdetectably labeled or further comprises a detectable label conjugated toit. Also provided is a hybridoma cell line that produces a monoclonalantibody of this invention. Compositions comprising one or more of theabove embodiments are further provided herein.

Also provided is a composition comprising the antibody and a carrier.Further provided is a biologically active fragment of the antibody, or acomposition comprising the antibody fragment. Suitable carriers aredefined supra.

Further provided is an antibody-peptide complex comprising, oralternatively consisting essentially of, or yet alternatively consistingof, the antibody and a polypeptide specifically bound to the antibody.In one aspect, the polypeptide is the chimeric polypeptide against whichthe antibody is raised.

This invention also provides an antibody capable of specifically forminga complex with Toso, which are useful in the therapeutic methods of thisinvention. Antibodies of the invention include, but are not limited tomouse, rat, and rabbit or human antibodies. Antibodies can be producedin cell culture, in phage, or in various animals, including but notlimited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs,sheep, dogs, cats, monkeys, chimpanzees, apes, etc. The antibodies arealso useful to identify and purify therapeutic polypeptides.

This invention also provides an antibody-peptide complex comprising, oralternatively consisting essentially of, or yet alternatively consistingof, antibodies described above and a polypeptide specifically bound tothe antibody. In one aspect the polypeptide is the polypeptide againstwhich the antibody was raised. In one aspect the antibody-peptidecomplex is an isolated complex. In a further aspect, the antibody of thecomplex is, but not limited to, a polyclonal antibody, a monoclonalantibody, a humanized antibody or an antibody derivative describedherein. Either or both of the antibody or peptide of theantibody-peptide complex can be detectably labeled or further comprisesa detectable label conjugated to it. In one aspect, the antibody-peptidecomplex of the invention can be used as a control or reference sample indiagnostic or screening assays.

Polyclonal antibodies of the invention can be generated usingconventional techniques known in the art and are well-described in theliterature. Several methodologies exist for production of polyclonalantibodies. For example, polyclonal antibodies are typically produced byimmunization of a suitable mammal such as, but not limited to, chickens,goats, guinea pigs, hamsters, horses, llamas, mice, rats, and rabbits.An antigen is injected into the mammal, which induces the B-lymphocytesto produce IgG immunoglobulins specific for the antigen. This IgG ispurified from the mammal's serum. Variations of this methodology includemodification of adjuvants, routes and site of administration, injectionvolumes per site and the number of sites per animal for optimalproduction and humane treatment of the animal. For example, adjuvantstypically are used to improve or enhance an immune response to antigens.Most adjuvants provide for an injection site antigen depot, which allowsfor a slow release of antigen into draining lymph nodes. Other adjuvantsinclude surfactants which promote concentration of protein antigenmolecules over a large surface area and immunostimulatory molecules.Non-limiting examples of adjuvants for polyclonal antibody generationinclude Freund's adjuvants, Ribi adjuvant system, and Titermax.Polyclonal antibodies can be generated using methods described in U.S.Pat. Nos. 7,279,559; 7,119,179; 7,060,800; 6,709,659; 6,656,746;6,322,788; 5,686,073; and 5,670,153, which are hereby incorporated byreference in their entirety for all purposes and in particular for allteachings related to antibodies.

The monoclonal antibodies of the invention can be generated usingconventional hybridoma techniques known in the art and well-described inthe literature. For example, a hybridoma is produced by fusing asuitable immortal cell line (e.g., a myeloma cell line such as, but notlimited to, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243,P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U397, MLA 144, ACT IV,MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144,NAMAIWA, NEURO 2A, CHO, PerC.6, YB2/O) or the like, or heteromyelomas,fusion products thereof, or any cell or fusion cell derived therefrom,or any other suitable cell line as known in the art (see, e.g.,www.atcc.org, www.lifetech.com., last accessed on Nov. 26, 2007, and thelike), with antibody producing cells, such as, but not limited to,isolated or cloned spleen, peripheral blood, lymph, tonsil, or otherimmune or B cell containing cells, or any other cells expressing heavyor light chain constant or variable or framework or CDR sequences,either as endogenous or heterologous nucleic acid, as recombinant orendogenous, viral, bacterial, algal, prokaryotic, amphibian, insect,reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination thereof. Antibodyproducing cells can also be obtained from the peripheral blood or,preferably the spleen or lymph nodes, of humans or other suitableanimals that have been immunized with the antigen of interest. Any othersuitable host cell can also be used for expressing-heterologous orendogenous nucleic acid encoding an antibody, specified fragment orvariant thereof, of the present invention. The fused cells (hybridomas)or recombinant cells can be isolated using selective culture conditionsor other suitable known methods, and cloned by limiting dilution or cellsorting, or other known methods.

In one embodiment, the antibodies described herein can be generatedusing a Multiple Antigenic Peptide (MAP) system. The MAP system utilizesa peptidyl core of three or seven radially branched lysine residues, onto which the antigen peptides of interest can be built using standardsolid-phase chemistry. The lysine core yields the MAP bearing about 4 to8 copies of the peptide epitope depending on the inner core thatgenerally accounts for less than 10% of total molecular weight. The MAPsystem does not require a carrier protein for conjugation. The highmolar ratio and dense packing of multiple copies of the antigenicepitope in a MAP has been shown to produce strong immunogenic response.This method is described in U.S. Pat. No. 5,229,490 and is hereinincorporated by reference in its entirety for all purposes and inparticular for all teachings related to the MAP system.

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, or the like, display library; e.g., asavailable from various commercial vendors such as Cambridge AntibodyTechnologies (Cambridgeshire, UK), MorphoSys (Martinsreid/Planegg,Del.), Biovation (Aberdeen, Scotland, UK) BioInvent (Lund, Sweden),using methods known in the art. See U.S. Pat. Nos. 4,704,692; 5,723,323;5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862, which are herebyincorporated by reference in their entirety for all purposes and inparticular for all teachings related to methods related to antibodies.Alternative methods rely upon immunization of transgenic animals (e.g.,SCID mice, Nguyen et al. (1997) Microbiol. Immunol. 41:901-907; Sandhuet al. (1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998)Immunol. 93:154-161 that are capable of producing a repertoire of humanantibodies, as known in the art and/or as described herein. Suchtechniques, include, but are not limited to, ribosome display (Hanes etal. (1997) Proc. Natl. Acad. Sci. USA 94:4937-4942; Hanes et al. (1998)Proc. Natl. Acad. Sci. USA 95:14130-14135); single cell antibodyproducing technologies (e.g., selected lymphocyte antibody method(“SLAM”) (U.S. Pat. No. 5,627,052, Wen et al. (1987) J. Immunol.17:887-892; Babcook et al. (1996) Proc. Natl. Acad. Sci. USA93:7843-7848); gel microdroplet and flow cytometry (Powell et al. (1990)Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass.); Gray et al.(1995) J. Imm. Meth. 182:155-163; and Kenny et al. (1995) Bio. Technol.13:787-790); B-cell selection (Steenbakkers et al. (1994) Molec. Biol.Reports 19:125-134, which are hereby incorporated by reference in theirentirety for all purposes and in particular for all teachings related tomethods for generating antibodies.

Antibody derivatives of the present invention can also be prepared bydelivering a polynucleotide encoding an antibody of this invention to asuitable host such as to provide transgenic animals or mammals, such asgoats, cows, horses, sheep, and the like, that produce such antibodiesin their milk. These methods are known in the art and are described forexample in U.S. Pat. Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992;5,994,616; 5,565,362; and 5,304,489, which are hereby incorporated byreference in their entirety for all purposes and in particular for allteachings related to generating antibodies.

The term “antibody derivative” includes post-translational modificationto linear polypeptide sequence of the antibody or fragment. For example,U.S. Pat. No. 6,602,684 B1, which is hereby incorporated by reference inits entirety for all purposes and in particular for all teachingsrelated to modifications of antibodies, describes a method for thegeneration of modified glycol-forms of antibodies, including wholeantibody molecules, antibody fragments, or fusion proteins that includea region equivalent to the Fc region of an immunoglobulin, havingenhanced Fc-mediated cellular toxicity, and glycoproteins so generated.

Antibody derivatives also can be prepared by delivering a polynucleotideof this invention to provide transgenic plants and cultured plant cells(e.g., but not limited to tobacco, maize, and duckweed) that producesuch antibodies, specified portions or variants in the plant parts or incells cultured therefrom. For example, Cramer et al. (1999) Curr. Top.Microbol. Immunol. 240:95-118 and references cited therein, describe theproduction of transgenic tobacco leaves expressing large amounts ofrecombinant proteins, e.g., using an inducible promoter. Transgenicmaize have been used to express mammalian proteins at commercialproduction levels, with biological activities equivalent to thoseproduced in other recombinant systems or purified from natural sources.See, e.g., Hood et al. (1999) Adv. Exp. Med. Biol. 464:127-147 andreferences cited therein. Antibody derivatives have also been producedin large amounts from transgenic plant seeds including antibodyfragments, such as single chain antibodies (scFv's), including tobaccoseeds and potato tubers. See, e.g., Conrad et al. (1998) Plant Mol.Biol. 38:101-109 and reference cited therein. Thus, antibodies of thepresent invention can also be produced using transgenic plants,according to know methods.

Antibody derivatives also can be produced, for example, by addingexogenous sequences to modify immunogenicity or reduce, enhance ormodify binding, affinity, on-rate, off-rate, avidity, specificity,half-life, or any other suitable characteristic. Generally part or allof the non-human or human CDR sequences are maintained while thenon-human sequences of the variable and constant regions are replacedwith human or other amino acids.

In general, the CDR residues (an example of an antibody fragment) aredirectly and most substantially involved in influencing antigen binding.Humanization or engineering of antibodies of the present invention canbe performed using any known method such as, but not limited to, thosedescribed in U.S. Pat. Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483;5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023;6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567,which are hereby incorporated by reference in their entirety for allpurposes and in particular for all teachings related to humanization orengineering of antibodies.

Techniques for making partially to fully human antibodies are known inthe art and any such techniques can be used. According to oneembodiment, fully human antibody sequences are made in a transgenicmouse which has been engineered to express human heavy and light chainantibody genes. Multiple strains of such transgenic mice have been madewhich can produce different classes of antibodies. B cells fromtransgenic mice which are producing a desirable antibody can be fused tomake hybridoma cell lines for continuous production of the desiredantibody. (See for example, Russel et al. (2000) Infection and Immunity68(4):1820-1826; Gallo et al. (2000) European J. of Immun. 30:534-540;Green (1999) J. of Immun. Methods 231:11-23; Yang et al. (1999A) J. ofLeukocyte Biology 66:401-410; Yang (1999B) Cancer Research59(6):1236-1243; Jakobovits (1998) Advanced Drug Delivery Reviews31:33-42; Green & Jakobovits (1998) J. Exp. Med. 188(3):483-495;Jakobovits (1998) Exp. Opin. Invest. Drugs 7(4):607-614; Tsuda et al.(1997) Genomics 42:413-421; Sherman-Gold (1997) Genetic Engineering News17(14); Mendez et al. (1997) Nature Genetics 15:146-156; Jakobovits(1996) Weir's Handbook of Experimental Immunology, The Integrated ImmuneSystem Vol. IV, 194.1-194.7; Jakobovits (1995) Current Opinion inBiotechnology 6:561-566; Mendez et al. (1995) Genomics 26:294-307;Jakobovits (1994) Current Biology 4(8):761-763; Arbones et al. (1994)Immunity 1(4):247-260; Jakobovits (1993) Nature 362(6417):255-258;Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA 90(6):2551-2555; andU.S. Pat. No. 6,075,181.)

The antibodies of this invention also can be modified to create chimericantibodies. Chimeric antibodies are those in which the various domainsof the antibodies' heavy and light chains are coded for by DNA from morethan one species. See, e.g., U.S. Pat. No. 4,816,567.

Alternatively, the antibodies of this invention can also be modified tocreate veneered antibodies. Veneered antibodies are those in which theexterior amino acid residues of the antibody of one species arejudiciously replaced or “veneered” with those of a second species sothat the antibodies of the first species will not be immunogenic in thesecond species thereby reducing the immunogenicity of the antibody.Since the antigenicity of a protein is primarily dependent on the natureof its surface, the immunogenicity of an antibody could be reduced byreplacing the exposed residues which differ from those usually found inanother mammalian species antibodies. This judicious replacement ofexterior residues should have little, or no, effect on the interiordomains, or on the interdomain contacts. Thus, ligand binding propertiesshould be unaffected as a consequence of alterations which are limitedto the variable region framework residues. The process is referred to as“veneering” since only the outer surface or skin of the antibody isaltered, the supporting residues remain undisturbed.

The procedure for “veneering” makes use of the available sequence datafor human antibody variable domains compiled by Kabat et al. (1987)Sequences of Proteins of Immunological Interest, 4th ed., Bethesda, Md.,National Institutes of Health, updates to this database, and otheraccessible U.S. and foreign databases (both nucleic acid and protein).Non-limiting examples of the methods used to generate veneeredantibodies include EP 519596; U.S. Pat. No. 6,797,492; and described inPadlan et al. (1991) Mol. Immunol. 28(4-5):489-498.

The term “antibody derivative” also includes “diabodies” which are smallantibody fragments with two antigen-binding sites, wherein fragmentscomprise a heavy chain variable domain (VH) connected to a light chainvariable domain (VL) in the same polypeptide chain. (See for example, EP404,097; WO 93/11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci.USA 90:6444-6448.) By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. (See also, U.S. Pat. No. 6,632,926 to Chen et al.which discloses antibody variants that have one or more amino acidsinserted into a hypervariable region of the parent antibody and abinding affinity for a target antigen which is at least about two foldstronger than the binding affinity of the parent antibody for theantigen.)

The term “antibody derivative” further includes “linear antibodies”. Theprocedure for making linear antibodies is known in the art and describedin Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, theseantibodies comprise a pair of tandem Fd segments (V_(H)-C_(H)1-VH-C_(H)1) which form a pair of antigen binding regions. Linearantibodies can be bispecific or monospecific.

The antibodies of this invention can be recovered and purified fromrecombinant cell cultures by known methods including, but not limitedto, protein A purification, ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe used for purification.

Antibodies of the present invention include naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a eukaryotic host, including, for example,yeast, higher plant, insect and mammalian cells, or alternatively from aprokaryotic cells as described above.

If a monoclonal antibody being tested binds with protein or polypeptide,then the antibody being tested and the antibodies provided by thehybridomas of this invention are equivalent. It also is possible todetermine without undue experimentation, whether an antibody has thesame specificity as the monoclonal antibody of this invention bydetermining whether the antibody being tested prevents a monoclonalantibody of this invention from binding the protein or polypeptide withwhich the monoclonal antibody is normally reactive. If the antibodybeing tested competes with the monoclonal antibody of the invention asshown by a decrease in binding by the monoclonal antibody of thisinvention, then it is likely that the two antibodies bind to the same ora closely related epitope. Alternatively, one can pre-incubate themonoclonal antibody of this invention with a protein with which it isnormally reactive, and determine if the monoclonal antibody being testedis inhibited in its ability to bind the antigen. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or a closely related, epitopic specificity as the monoclonalantibody of this invention.

The term “antibody” also is intended to include antibodies of allisotypes. Particular isotypes of a monoclonal antibody can be preparedeither directly by selecting from the initial fusion, or preparedsecondarily, from a parental hybridoma secreting a monoclonal antibodyof different isotype by using the sib selection technique to isolateclass switch variants using the procedure described in Steplewski et al.(1985) Proc. Natl. Acad. Sci. USA 82:8653 or Spira et al. (1984) J.Immunol. Methods 74:307.

The isolation of other hybridomas secreting monoclonal antibodies withthe specificity of the monoclonal antibodies of the invention can alsobe accomplished by one of ordinary skill in the art by producinganti-idiotypic antibodies. Herlyn et al. (1986) Science 232:100. Ananti-idiotypic antibody is an antibody which recognizes uniquedeterminants present on the monoclonal antibody produced by thehybridoma of interest.

Idiotypic identity between monoclonal antibodies of two hybridomasdemonstrates that the two monoclonal antibodies are the same withrespect to their recognition of the same epitopic determinant. Thus, byusing antibodies to the epitopic determinants on a monoclonal antibodyit is possible to identify other hybridomas expressing monoclonalantibodies of the same epitopic specificity.

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is themirror image of the epitope bound by the first monoclonal antibody.Thus, in this instance, the anti-idiotypic monoclonal antibody could beused for immunization for production of these antibodies.

In some aspects of this invention, it will be useful to detectably ortherapeutically label the antibody. Methods for conjugating antibodiesto these agents are known in the art. For the purpose of illustrationonly, antibodies can be labeled with a detectable moiety such as aradioactive atom, a chromophore, a fluorophore, or the like. Suchlabeled antibodies can be used for diagnostic techniques, either invivo, or in an isolated test sample.

The coupling of antibodies to low molecular weight haptens can increasethe sensitivity of the antibody in an assay. The haptens can then bespecifically detected by means of a second reaction. For example, it iscommon to use haptens such as biotin, which reacts avidin, ordinitrophenol, pyridoxal, and fluorescein, which can react with specificanti-hapten antibodies. See, Harlow & Lane (1988) supra.

The antibodies of the invention also can be bound to many differentcarriers. Thus, this invention also provides compositions containing theantibodies and another substance, active or inert. Examples ofwell-known carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindingmonoclonal antibodies, or will be able to ascertain such, using routineexperimentation.

In certain embodiments, antibodies of the invention include mutations inthe constant region that improve pharmacokinetic properties of theantibodies as compared to antibodies without such mutations. Suchantibodies will in certain embodiments include an Fc domain that isderived from human IgG1 at the C-terminus, which in yet furtherembodiments include mutations that diminish or ablate antibody-dependentand complement dependent cytotoxicity. In a still further embodiment,such mutations include one or more of the following mutations singly orin any combination: E233P; L234V; L235A; ΔG236; A327G; A330S; P331S. Ina yet further embodiment, the Fc domain comprises a sequence accordingto SEQ ID NO: 3, which is shown in FIG. 10. In a still furtherembodiment, the Fc domain comprises a sequence with a sequence identityof about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% and 100% identity to SEQ ID NO: 3.

In further embodiments, one or more amino acid modifications are made inone or more of the CDRs of the antibody. In general, only 1 or 2 or 3amino acids are substituted in any single CDR, and generally no morethan from 4, 5, 6, 7, 8 9 or 10 changes are made within a set of CDRs.However, it should be appreciated that any combination of nosubstitutions, 1, 2 or 3 substitutions in any CDR can be independentlyand optionally combined with any other substitution.

In some cases, amino acid modifications in the CDRs are referred to as“affinity maturation”. An “affinity matured” antibody is one having oneor more alteration(s) in one or more CDRs which results in animprovement in the affinity of the antibody for antigen, compared to aparent antibody which does not possess those alteration(s). In somecases, although rare, it may be desirable to decrease the affinity of anantibody to its antigen, but this is generally not preferred.

Affinity maturation can be conducted to increase the binding affinity ofthe antibody for the antigen by at least about 10% to 50-100-150% ormore, or from 1 to 5 fold as compared to the “parent” antibody.Preferred affinity matured antibodies will have nanomolar or evenpicomolar affinities for the target antigen. Affinity matured antibodiesare produced by known procedures. See, for example, Marks et al., 1992,Biotechnology 10:779-783 that describes affinity maturation by variableheavy chain (VH) and variable light chain (VL) domain shuffling. Randommutagenesis of CDR and/or framework residues is described in: Barbas, etal. 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813; Shier et al., 1995,Gene 169:147-155; Yelton et al., 1995, J. Immunol. 155:1994-2004;Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al,1992, J. Mol. Biol. 226:889-896, for example.

Alternatively, amino acid modifications can be made in one or more ofthe CDRs of the antibodies of the invention that are “silent”, e.g. thatdo not significantly alter the affinity of the antibody for the antigen.These can be made for a number of reasons, including optimizingexpression (as can be done for the nucleic acids encoding the antibodiesof the invention).

Thus, included within the definition of the CDRs and antibodies of theinvention are variant CDRs and antibodies; that is, the antibodies ofthe invention can include amino acid modifications in one or more of theCDRs of Ab79 and Ab19. In addition, as outlined below, amino acidmodifications can also independently and optionally be made in anyregion outside the CDRs, including framework and constant regions.

In some embodiments, the antibodies of the invention are conjugated withdrugs to form antibody-drug conjugates (ADCs). In general, ADCs are usedin oncology applications, where the use of antibody-drug conjugates forthe local delivery of cytotoxic or cytostatic agents allows for thetargeted delivery of the drug moiety to tumors, which can allow higherefficacy, lower toxicity, etc. An overview of this technology isprovided in Ducry et al., Bioconjugate Chem., 21:5-13 (2010), Carter etal., Cancer J. 14(3):154 (2008) and Senter, Current Opin. Chem. Biol.13:235-244 (2009), all of which are hereby incorporated by reference intheir entirety for all purposes and in particular for all teachingsrelated to antibody drug conjugates.

Thus, in some embodiments, the invention provides Toso antibodiesconjugated to drugs. Generally, conjugation is done by covalentattachment to the antibody and generally relies on a linker, often apeptide linkage (which, as is known in the art, may be designed to besensitive to cleavage by proteases at the target site or not). Inaddition, as described above, linkage of the linker-drug unit (LU-D) canbe done by attachment to cysteines within the antibody. As will beappreciated by those in the art, the number of drug moieties perantibody can change, depending on the conditions of the reaction, andcan vary from 1:1 to 10:1 drug:antibody. As will be appreciated by thosein the art, the actual number is an average.

The drug of the ADC can be any number of agents, including but notlimited to cytotoxic agents such as chemotherapeutic agents, growthinhibitory agents, toxins (for example, an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (that is, a radioconjugate) are provided. In otherembodiments, the invention further provides methods of using the ADCs.

Drugs for use in antibody-drug conjugates of the present inventioninclude cytotoxic drugs, particularly those which are used for cancertherapy. Such drugs include, in general, DNA damaging agents,anti-metabolites, natural products and their analogs. Exemplary classesof cytotoxic agents include the enzyme inhibitors such as dihydrofolatereductase inhibitors, and thymidylate synthase inhibitors, DNAintercalators, DNA cleavers, topoisomerase inhibitors, the anthracyclinefamily of drugs, the vinca drugs, the mitomycins, the bleomycins, thecytotoxic nucleosides, the pteridine family of drugs, diynenes, thepodophyllotoxins, dolastatins, maytansinoids, differentiation inducers,and taxols.

Members of these classes include, for example, methotrexate,methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine,cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin,daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin,aminopterin, tallysomycin, podophyllotoxin and podophyllotoxinderivatives such as etoposide or etoposide phosphate, vinblastine,vincristine, vindesine, taxanes including taxol, taxotere retinoic acid,butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin,esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin,camptothecin, maytansinoids (including DM1), monomethylauristatin E(MMAE), monomethylauristatin F (MMAF), and maytansinoids (DM4) and theiranalogues.

Toxins may be used as antibody-toxin conjugates and include bacterialtoxins such as diphtheria toxin, plant toxins such as ricin, smallmolecule toxins such as geldanamycin (Mandler et al (2000) J. Nat.Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342).Toxins may exert their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.

Conjugates of a Toso antibody and one or more small molecule toxins,such as a maytansinoids, dolastatins, auristatins, a trichothecene,calicheamicin, and CC1065, and the derivatives of these toxins that havetoxin activity, are contemplated.

In accordance with any of the above, another type of modification thatcan be made to antibodies of the invention is alterations inglycosylation. In another embodiment, the antibodies disclosed hereincan be modified to include one or more engineered glycoforms. By“engineered glycoform” as used herein is meant a carbohydratecomposition that is covalently attached to the antibody, wherein saidcarbohydrate composition differs chemically from that of a parentantibody. Engineered glycoforms may be useful for a variety of purposes,including but not limited to enhancing or reducing effector function. Anexemplary form of engineered glycoform is afucosylation, which has beenshown to be correlated to an increase in ADCC function, presumablythrough tighter binding to the FcγRIIIa receptor. In this context,“afucosylation” means that the majority of the antibody produced in thehost cells is substantially devoid of fucose, e.g. 90-95-98% of thegenerated antibodies do not have appreciable fucose as a component ofthe carbohydrate moiety of the antibody (generally attached at N297 inthe Fc region). Defined functionally, afucosylated antibodies generallyexhibit at least a 50% or higher affinity to the FcγRIIIa receptor.

Engineered glycoforms may be generated by a variety of methods known inthe art (Umana et al., 1999, Nat Biotechnol 17:176-180; Davies et al.,2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; U.S.Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929;PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCT WO02/30954A1, all entirely incorporated by reference in their entirety forall purposes and in particular for all teachings related to engineeredglycoforms. Many of these techniques are based on controlling the levelof fucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region, for example by expressing an IgG in variousorganisms or cell lines, engineered or otherwise (for example Lec-13 CHOcells or rat hybridoma YB2/0 cells, by regulating enzymes involved inthe glycosylation pathway (for example FUT8 [α1,6-fucosyltransferase]and/or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or bymodifying carbohydrate(s) after the IgG has been expressed. For example,the “sugar engineered antibody” or “SEA technology” of Seattle Geneticsfunctions by adding modified saccharides that inhibit fucosylationduring production; see for example 20090317869, hereby incorporated byreference in its entirety. Engineered glycoform typically refers to thedifferent carbohydrate or oligosaccharide; thus an antibody can includean engineered glycoform.

Alternatively, engineered glycoform may refer to a variant thatcomprises the different carbohydrate or oligosaccharide. As is known inthe art, glycosylation patterns can depend on both the sequence of theprotein (e.g., the presence or absence of particular glycosylation aminoacid residues, discussed below), or the host cell or organism in whichthe protein is produced. Particular expression systems are known in theart and discussed herein.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to an antibody (or to any otherpolypeptide, such as the soluble Toso protein discussed above) isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tri-peptide sequences(for N-linked glycosylation sites). The alteration may also be made bythe addition of, or substitution by, one or more serine or threonineresidues to the starting sequence (for O-linked glycosylation sites).For ease, the antibody amino acid sequence is preferably altered throughchanges at the DNA level, particularly by mutating the DNA encoding thetarget polypeptide at preselected bases such that codons are generatedthat will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on anantibody or another protein is by chemical or enzymatic coupling ofglycosides to the protein. These procedures are advantageous in thatthey do not require production of the protein in a host cell that hasglycosylation capabilities for N- and O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine, (b) free carboxyl groups, (c) free sulfhydryl groups suchas those of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 and in Aplin andWriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306, both entirelyincorporated by reference herein in their entirety for all purposes andin particular for all teachings related to coupling carbohydratemoieties to proteins.

Removal of carbohydrate moieties present on the starting antibody (e.g.post-translationally) may be accomplished chemically or enzymatically.Chemical deglycosylation requires exposure of the protein to thecompound trifluoromethanesulfonic acid, or an equivalent compound. Thistreatment results in the cleavage of most or all sugars except thelinking sugar (N-acetylglucosamine or N-acetylgalactosamine), whileleaving the polypeptide intact. Chemical deglycosylation is described byHakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge etal., 1981, Anal. Biochem. 118:131, both entirely incorporated byreference. Enzymatic cleavage of carbohydrate moieties on polypeptidescan be achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., 1987, Meth. Enzymol. 138:350, entirelyincorporated by reference. Glycosylation at potential glycosylationsites may be prevented by the use of the compound tunicamycin asdescribed by Duskin et al., 1982, J. Biol. Chem. 257:3105, entirelyincorporated by reference. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of the antibody comprises linkingthe antibody to various non proteinaceous polymers, including, but notlimited to, various polyols such as polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in, for example,2005-2006 PEG Catalog from Nektar Therapeutics (available at the Nektarwebsite) U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337, all entirely incorporated by reference for allpurposes and in particular for all teachings related to linkingantibodies to polymers. In addition, as is known in the art, amino acidsubstitutions may be made in various positions within the antibody tofacilitate the addition of polymers such as PEG. See for example, U.S.Publication No. 2005/0114037A1, entirely incorporated by reference.

The present invention further includes the nucleic acids encoding theToso antibodies of the invention. In the case where both a heavy andlight chain constant domains are included in the antibody, generallythese are made using nucleic acids encoding each, that are combined intostandard host cells (e.g. CHO cells, etc.) to produce the tetramericstructure of the antibody. If only one constant domain is being made,only a single nucleic acid will be used.

Formulations of the antibodies used in accordance with the presentinvention can be prepared for storage by mixing an antibody having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. [1980]), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulations of the invention may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. For example, it may be desirable to provideantibodies with other specificities. Alternatively, or in addition, thecomposition may comprise a cytotoxic agent, cytokine, growth inhibitoryagent and/or small molecule antagonist. Such molecules are suitablypresent in combination in amounts that are effective for the purposeintended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration should besterile, or nearly so. This is readily accomplished by filtrationthrough sterile filtration membranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gammaethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT®(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

When encapsulated antibodies remain in the body for a long time, theymay denature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism has been shown to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

IV. Methods of Modulating Toso Activity

In one aspect, the present invention is directed to methods ofmodulating Toso activity. In one embodiment, methods of modulating Tosoactivity comprise inhibiting Toso activity. In other embodiments,methods of modulating Toso activity comprise increasing Toso activity.

In some embodiments, methods of the present invention involve directlymodulating Toso activity. In an exemplary embodiment, such methodsinclude applying an agent that binds to Toso, such as an antibody.

In other embodiments, Toso activity is modulated indirectly, for exampleby binding cognate ligands of Toso. In an exemplary embodiment, Tosoactivity is modulated by administering a soluble Toso protein.

In further embodiments, Toso activity is modulated by a combination ofmechanisms, for example by administering a composition comprising anagent that binds to Toso in combination with a composition comprising anagent that binds to cognate ligands of Toso. In an exemplary embodiment,such a combination may include without limitation a Toso antibody and asoluble Toso protein.

As will be appreciated, methods of modulating Toso activity can includethe use of any of the compositions described herein in any combination,including any one or more of SEQ ID NOs. 1-25 as well as any variants ormodifications thereof as described herein.

V. Methods of Treating Disorders

In one aspect and in accordance with any of the above, the presentinvention provides methods of treating disorders by treating subjects inneed thereof with a composition that modulates Toso activity, includingwithout limitation a soluble Toso protein or an antibody to Toso.

In a specific embodiment and in accordance with any of the above, thepresent invention provides methods of treating disorders by treatingsubjects in need thereof with a composition that includes a soluble Tosoprotein. Without being limited by theory, one potential mechanism bywhich the soluble Toso protein is an effective treatment for thesedisorders is through modulating Toso activity. In certain embodiments,methods of treating disorders in accordance with the present inventionincludes administering a therapeutically effective amount of any of thesoluble Toso proteins described herein, including soluble Toso proteinscomprising any one or more of SEQ ID NOs: 1-25 or any variants thereof.In further embodiments, the soluble Toso proteins used to treatdisorders, including diabetes, multiple sclerosis, asthma, and cancerinclude a polypeptide with about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% identity to any one of SEQ ID NOs: 1-25.such polypeptides may further be modified in accordance with the methodsdescribed herein, including chemical modifications, for the treatment ofany of the disorders described herein.

In further aspects, the present invention is directed to methods oftreating disorders and diseases by administering a soluble Toso protein(or a variant thereof) to a subject. Soluble Toso proteins of theinvention can be used to treat subjects suffering from withoutlimitation: an autoimmune disorder (including without limitation Type 1diabetes, multiple sclerosis, or rheumatoid arthritis), Type 2 diabetes,asthma, allergy chronic obstructive pulmonary disease (“COPD”),hyper-IgM syndrome, lupus, cancer, or a neutrophilia-associated disorder(including without limitation neutropenia, severe congenitalneutropenia, cyclical neutropenia, antibody mediated neutropenia,reticular dysgenesis, leukocyte adhesion deficiency, familiarmyeloproliferative disease, chronic myelogenous leukemia, familiar coldurticaria and leukocytosis, and chronic granulomatous disease). As willbe appreciated, any of the soluble Toso proteins described herein,singly or in any combination, can be used to treat any of thesedisorders or diseases.

In further embodiments, a pharmaceutically acceptable amount of asoluble Toso protein is administered to a subject in need thereof totreat any of the disorders discussed herein. In some embodiments, thesoluble Toso protein administered to the subject includes anextracellular Toso domain and/or an Fc domain and/or a signal sequenceand/or a flexible linker. In still further embodiments, the soluble Tosoprotein comprises a sequence according to any one of SEQ ID NOs: 1-25.Combinations of any one of SEQ ID NOs.:1-25 may also be used, either asseparate polypeptides or together as fusion proteins, to treat any ofthe disorders discussed herein. Such polypeptides may also be furthermodified, including chemically modified, in accordance with thedescription herein to treat such disorders. In still furtherembodiments, the soluble Toso protein used to treat any of the disordersdescribed herein has a sequence comprising SEQ ID NO: 5, which is shownin FIG. 8. In yet further embodiments, the soluble Toso proteinadministered to a subject for the treatment of any of the disordersdiscussed herein has a sequence with at least about 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO.5. In further embodiments, the soluble Toso protein administered to asubject for the treatment of any of the disorders discussed herein has asequence with about 75-99%, 80-98%, 85-97%, 90-96%, 91-99%, 92-98%,93-97%, 94-96% identity to SEQ ID NO: 5. In still further embodiments,the soluble Toso protein administered to the subject for the treatmentof any of the disorders discussed herein comprises SEQ ID NO. 5 with 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20amino acid substitutions. In yet further embodiments, the soluble Tosoprotein administered to the subject for the treatment of diabetescomprises SEQ ID NO: 5 with 1-30, 2-25, 3-20, 4-15, 5-10, 6-9, 7-8 aminoacid substitutions. In further exemplary embodiments, about 12.1, 12.2,12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.1, 13.2, 13.3, 13.4, 13.5,13.6, 13.7, 13.8, 13.9, 14 mg of the soluble Toso protein isadministered to the subject for a therapeutic effect. As will beappreciated, the amount of soluble Toso protein can be ascertained basedon animal studies using the widely accepted Body Surface Area (BSA)normalization method used for the conversion of dosages fromexperimental animals to humans. (see Reagan-Shaw, S., Nihal, M., andAdmad, N. Dose translation from animal to human studies revisited. 2007.The Faseb Journal).

In specific embodiments, methods and compositions of the invention areused to treat subjects at risk for or that have Type 1 or Type 2diabetes. In further embodiments, a pharmaceutically acceptable amountof a soluble Toso protein is administered to a subject in need thereof.In some embodiments, the soluble Toso protein administered to thesubject to treat diabetes includes an extracellular Toso domain and/oran Fc domain and/or a signal sequence and/or a linker. In still furtherembodiments, the soluble Toso protein comprises a sequence according toany one of SEQ ID NOs: 1-25 or any of the variants of SEQ ID NOs: 1-25discussed herein. In yet further embodiments, the soluble Toso proteinadministered to a subject for the treatment of diabetes includes asequence with at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% identity to any one of SEQ ID NOs: 1-25.Combinations of any one of SEQ ID NOs.:1-25 may also be used, either asseparate polypeptides or together as fusion proteins, to treat diabetes.Such polypeptides may also be further modified, including chemicallymodified, in accordance with the description herein to treat diabetes.In still further embodiments, the soluble Toso protein used to treat asubject for diabetes has a sequence comprising SEQ ID NO: 5, which isshown in FIG. 8. In yet further embodiments, the soluble Toso proteinadministered to a subject for the treatment of diabetes has a sequencewith at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identity to SEQ ID NO. 5. In further embodiments, thesoluble Toso protein administered to a subject for the treatment ofdiabetes has a sequence with about 75-99%, 80-98%, 85-97%, 90-96%,91-99%, 92-98%, 93-97%, 94-96% identity to SEQ ID NO: 5. In stillfurther embodiments, the soluble Toso protein administered to thesubject for the treatment of diabetes comprises SEQ ID NO. 5 with 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aminoacid substitutions. In yet further embodiments, the soluble Toso proteinadministered to the subject for the treatment of diabetes comprises SEQID NO: 5 with 1-30, 2-25, 3-20, 4-15, 5-10, 6-9, 7-8 amino acidsubstitutions. In still further embodiments, treatment with a solubleToso protein in accordance with any of the compositions described hereinserves to improve glucose tolerance in a subject, and thereby treatdiabetes. In exemplary embodiments, about 10-20, 11-19, 12-18, 13-17,14-16 mg of the soluble Toso protein is administered to the subject fora therapeutic effect. In further exemplary embodiments, about 10, 10.1,10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3,11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5,12.6, 12.7, 12.8, 12.9, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8,13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15,15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16 mg of thesoluble Toso protein is administered to the subject for a therapeuticeffect. As will be appreciated, the amount of soluble Toso protein canbe ascertained based on animal studies using the widely accepted BodySurface Area (BSA) normalization method used for the conversion ofdosages from experimental animals to humans. (see Reagan-Shaw, S.,Nihal, M., and Admad, N. Dose translation from animal to human studiesrevisited. 2007. The Faseb Journal). For the experiments described infurther detail herein, 50 μg doses were used in the disease models,which would approximately be 2.5 mg/kg in a 20 g mouse. Using the BSAconversion, this would be 0.2027 mg/kg or 7.5 mg/m², or approximately12.2 mg for a 60 kg adult.

In other embodiments, methods and compositions of the invention are usedto treat subjects at risk for or that have multiple sclerosis. Infurther embodiments, a pharmaceutically acceptable amount of a solubleToso protein is administered to a subject in need thereof for thetreatment or amelioration of multiple sclerosis. In some embodiments,the soluble Toso protein administered to the subject includes and Fcdomain and/or a flexible linker. In some embodiments, the soluble Tosoprotein administered to the subject includes an extracellular Tosodomain and/or an Fc domain and/or a signal sequence and/or a linker. Instill further embodiments, the soluble Toso protein comprises a sequenceaccording to any one of SEQ ID NOs: 1-24. Combinations of any one of SEQID NOs.:1-25 may also be used, either as separate polypeptides ortogether as fusion proteins, to treat multiple sclerosis. Suchpolypeptides may also be further modified, including chemicallymodified, in accordance with the description herein to treat multiplesclerosis. In still further embodiments, the soluble Toso protein has asequence comprising SEQ ID NO: 5, which is shown in FIG. 8. In yetfurther embodiments, the soluble Toso protein administered to a subjectfor the treatment of multiple sclerosis has a sequence with at leastabout 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% identity to SEQ ID NO. 5. In further embodiments, the soluble Tosoprotein administered to a subject for the treatment of multiplesclerosis has a sequence with about 75-99%, 80-98%, 85-97%, 90-96%,91-99%, 92-98%, 93-97%, 94-96% identity to SEQ ID NO: 5. In stillfurther embodiments, the soluble Toso protein administered to thesubject for the treatment of multiple sclerosis comprises SEQ ID NO. 5with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20 amino acid substitutions. In yet further embodiments, the solubleToso protein administered to the subject for the treatment of multiplesclerosis comprises SEQ ID NO: 5 with 1-30, 2-25, 3-20, 4-15, 5-10, 6-9,7-8 amino acid substitutions. In still further embodiments, treatmentwith a soluble Toso protein in accordance with any of the compositionsdescribed herein serves to delay the progression of multiple sclerosis.In exemplary embodiments, about 10-20, 11-19, 12-18, 13-17, 14-16 mg ofthe soluble Toso protein is administered to the subject for atherapeutic effect. In further exemplary embodiments, about 10, 10.1,10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3,11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5,12.6, 12.7, 12.8, 12.9, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8,13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15,15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16 mg of thesoluble Toso protein is administered to the subject for a therapeuticeffect. As will be appreciated, the amount of soluble Toso protein canbe ascertained based on animal studies using the widely accepted BodySurface Area (BSA) normalization method used for the conversion ofdosages from experimental animals to humans. (see Reagan-Shaw, S.,Nihal, M., and Admad, N. Dose translation from animal to human studiesrevisited. 2007. The Faseb Journal). For the experiments described infurther detail herein, 50 μg doses were used in the disease models,which would approximately be 2.5 mg/kg in a 20 g mouse. Using the BSAconversion, this would be 0.2027 mg/kg or 7.5 mg/m², or approximately12.2 mg for a 60 kg adult.

In other embodiments, methods and compositions of the invention are usedto treat subjects at risk for or that have arthritis. In furtherembodiments, a pharmaceutically acceptable amount of a soluble Tosoprotein is administered to a subject in need thereof for the treatmentor prevention of arthritis. In some embodiments, the soluble Tosoprotein administered to the subject includes and Fc domain and/or alinker. In some embodiments, the soluble Toso protein administered tothe subject includes an extracellular Toso domain and/or an Fc domainand/or a signal sequence and/or a linker. In still further embodiments,the soluble Toso protein comprises a sequence according to any one ofSEQ ID NOs: 1-25 Combinations of any one of SEQ ID NOs:1-25 may also beused, either as separate polypeptides or together as fusion proteins, totreat arthritis. Such polypeptides may also be further modified,including chemically modified, in accordance with the description hereinto treat arthritis. In still further embodiments, the soluble Tosoprotein has a sequence comprising SEQ ID NO: 5, which is shown in FIG.8. In yet further embodiments, the soluble Toso protein administered toa subject for the treatment of arthritis has a sequence with at leastabout 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% identity to SEQ ID NO. 5. In further embodiments, the soluble Tosoprotein administered to a subject for the treatment of arthritis has asequence with about 75-99%, 80-98%, 85-97%, 90-96%, 91-99%, 92-98%,93-97%, 94-96% identity to SEQ ID NO: 5. In still further embodiments,the soluble Toso protein administered to the subject for the treatmentof arthritis comprises SEQ ID NO. 5 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions. Inyet further embodiments, the soluble Toso protein administered to thesubject for the treatment of arthritis comprises SEQ ID NO: 5 with 1-30,2-25, 3-20, 4-15, 5-10, 6-9, 7-8 amino acid substitutions. In stillfurther embodiments, treatment with a soluble Toso protein in accordancewith any of the compositions described herein serves to prevent theincidence of arthritis or protect against the severity of arthritis.

In specific embodiments, methods and compositions of the invention areused to treat subjects at risk for or that have asthma. In furtherembodiments, a pharmaceutically acceptable amount of a soluble Tosoprotein is administered to a subject in need thereof. In someembodiments, the soluble Toso protein administered to the subject totreat asthma includes an extracellular Toso domain and/or an Fc domainand/or a signal sequence and/or a linker. In still further embodiments,the soluble Toso protein comprises a sequence according to any one ofSEQ ID NOs: 1-25 or any of the variants of SEQ ID NOs: 1-25 discussedherein. In yet further embodiments, the soluble Toso proteinadministered to a subject for the treatment of asthma includes asequence with at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% identity to any one of SEQ ID NOs: 1-25.Combinations of any one of SEQ ID NOs.:1-25 may also be used, either asseparate polypeptides or together as fusion proteins, to treat asthma.Such polypeptides may also be further modified, including chemicallymodified, in accordance with the description herein to treat asthma. Instill further embodiments, the soluble Toso protein used to treat asubject for asthma has a sequence comprising SEQ ID NO: 5, which isshown in FIG. 8. In yet further embodiments, the soluble Toso proteinadministered to a subject for the treatment of asthma has a sequencewith at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identity to SEQ ID NO. 5. In further embodiments, thesoluble Toso protein administered to a subject for the treatment ofasthma has a sequence with about 75-99%, 80-98%, 85-97%, 90-96%, 91-99%,92-98%, 93-97%, 94-96% identity to SEQ ID NO: 5. In still furtherembodiments, the soluble Toso protein administered to the subject forthe treatment of asthma comprises SEQ ID NO. 5 with 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsubstitutions. In yet further embodiments, the soluble Toso proteinadministered to the subject for the treatment of asthma comprises SEQ IDNO: 5 with 1-30, 2-25, 3-20, 4-15, 5-10, 6-9, 7-8 amino acidsubstitutions. In still further embodiments, treatment with a solubleToso protein in accordance with any of the compositions described hereinserves to improve glucose tolerance in a subject, and thereby treatasthma. In exemplary embodiments, about 10-20, 11-19, 12-18, 13-17,14-16 mg of the soluble Toso protein is administered to the subject fora therapeutic effect. In further exemplary embodiments, about 10, 10.1,10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3,11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5,12.6, 12.7, 12.8, 12.9, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8,13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15,15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16 mg of thesoluble Toso protein is administered to the subject for a therapeuticeffect. As will be appreciated, the amount of soluble Toso protein canbe ascertained based on animal studies using the widely accepted BodySurface Area (BSA) normalization method used for the conversion ofdosages from experimental animals to humans. (see Reagan-Shaw, S.,Nihal, M., and Admad, N. Dose translation from animal to human studiesrevisited. 2007. The Faseb Journal). For the experiments described infurther detail herein, 50 μg doses were used in the disease models,which would approximately be 2.5 mg/kg in a 20 g mouse. Using the BSAconversion, this would be 0.2027 mg/kg or 7.5 mg/m², or approximately12.2 mg for a 60 kg adult.

In further embodiments, the soluble Toso proteins described herein,including the soluble Toso proteins comprising any one or more of thepolypeptides of SEQ ID NOs. 1-25, is in the form for use as amedicament. In further embodiments, the present invention provides asoluble Toso protein comprising any one or more (or a portion of any oneor more) of the polypeptides of SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 foruse as a medicament. In still further embodiments, the present inventionprovides methods for the use of a soluble Toso protein comprising anyone or more of the polypeptides of SEQ ID NOs. 1-25 for treating any oneof the following disorders: an autoimmune disorder (including withoutlimitation Type 1 or Type 2 diabetes, multiple sclerosis, or rheumatoidarthritis), asthma, allergy chronic obstructive pulmonary disease(“COPD”), hyper-IgM syndrome, lupus, cancer, or aneutrophilia-associated disorder (including without limitationneutropenia, severe congenital neutropenia, cyclical neutropenia,antibody mediated neutropenia, reticular dysgenesis, leukocyte adhesiondeficiency, familiar myeloproliferative disease, chronic myelogenousleukemia, familiar cold urticaria and leukocytosis, and chronicgranulomatous disease).

As discussed above, in some embodiments, soluble Toso proteins of theinvention are used to treat cancer. In further embodiments, methods oftreating cancer in accordance with the invention include methods ofinhibiting tumor invasion and/or metastasis by modulating Toso activity.In exemplary embodiments, compositions of the invention are used totreat any one of the group of an adenocarcinoma, a leukemia, a lymphoma,a melanoma, a myeloma, a sarcoma or a teratocarcinoma in subjects inneed thereof. In further embodiments, compositions of the invention areused to treat subjects suffering from a cancer in one or more of adrenalgland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid or uterus.

Example 1 Toso Plays a Role in the Pathogenesis of Arthritis

Toso^(−/−) and wild type mice were injected with Type II chickencollagen in Complete Freund's adjuvant subcutaneously and monitored thedisease progression over time. Animals exposed to Type II collagendevelop disease that has similar immunologic, pathologic andhistological features as human Rheumatoid Arthritis (RA). Jointinflammation, as measured by the change in ankle thickness using adigital caliper, was significantly reduced in Toso^(−/−) mice (FIG. 1A).In addition, disease severity was scored from 0-4 for each joint basedon visible swelling and mobility. Toso^(−/−) mice had drasticallyreduced clinical scores compared to wild type controls (FIG. 1B). Flowcytometric analysis of lymphocyte populations in the draining lymph nodeshowed a significant reduction in B220⁺ B cells (FIG. 1C). These datasuggest that Toso has a significant role in the pathogenesis ofarthritis.

Example 2 Treatment with Toso-Fc Protects Against Arthritis

This example shows that treatment with Toso-Fc (SEQ ID NO: 5) protectsagainst arthritis. Arthritis susceptible mice, DBA1, were pre-dosed with50 μg Toso-Fc I.P, immunized with Type II collagen in Complete Freund'sAdjuvant to induce disease, and then treated with Toso-Fc three timesper week over the course of the experiment. Toso-Fc treated mice wereprotected against both the severity and incidence of disease, suggestingthat Toso-Fc administration may be useful in the management ofarthritis. For example, FIG. 24A shows that the additive arthritis scorewas negligible in the Toso-Fc treated mice as compared to the controlmice (treated only with the vehicle). Similarly, FIG. 24B also showsthat the percent incidence of arthritis was negligible in the Toso-Fctreated mice as compared to control. Clinical symptoms of arthritis wereassessed as follows; 0=normal, 1=slight swelling and/or erythema,2=pronounced swelling, 3=ankylosis. The individual limb scores for eachmouse were added, giving a maximum disease score of 12. Incidence ofdisease was noted when mice were observed to have a disease score of 1in a limb.

FIG. 25 provides further data showing the recall response(proliferation) of splenocytes from Toso-Fc and vehicle treated micestimulated with Collagen. As shown in the figure, splenocytes from micetreated with Toso-Fc show a reduced proliferative response thansplenocytes from vehicle treated controls.

Example 3 Toso-Fc is Effective Against Arthritis Once the Disease isEstablished

As shown above, prophylactic administration of Toso-Fc ameliorateddisease symptoms in a murine model of rheumatoid arthritis. This exampleshows that the administration of the Toso-Fc is also effective in atherapeutic context (i.e., when disease is already established.

Female DBA.1 mice (6 to 8 weeks of age) were randomly assigned into 2groups, one to receive vehicle treatment, and the other to receiveToso-Fc when disease was observed. All mice were inoculated withCollagen to induce disease. Initiation of disease symptoms were observedon day 43 after immunization. The animals were treated with vehiclecontrol or Toso-Fc on day 52, as indicated by the arrow on the plots(FIG. 26A and FIG. 26B). After day 57, therapeutic administration ofToso-Fc dramatically reduced total disease score and arthritis incidence(see FIG. 26A and FIG. 26B). These data indicate that Toso-Fc caneffectively manage arthritis symptoms when disease has already beenestablished, and can thus be effective in a therapeutic as well as aprophylactic context.

Example 4 Toso Plays a Role in the Development of Asthma

Asthma is an allergic disorder characterized by aberrant TH2 activation,IgE production, bronchial hyperreactivity, and leukocyte extravasationinto the bronchial mucosa. Eosinophillia is a hallmark of allergicasthma and is thought to be a critical player in inflammation. Thepathologic manifestations of asthma cause the lungs to constrict,leading to wheezing, shortness of breath, chest tightness, and coughing.

An OVA induced asthma model was conducted on Toso−/− and wild type mice(FIG. 2A). OVA (10 mg/kg) were delivered intraperitoneally on days 0, 7,and 14. Mice then received aerosolled OVA (1 mg/ml) for 30 minutes ondays 21, 22, and 23, and sacrificed 2 days later. Toso−/− mice had adrastic reduction in eosinophil migration into the broncheoalveolarspace as assessed by DifQuik staining (FIG. 2B). Based on morphology,eosinophils were quantified by counting at least 200 leukocytes perslide (FIG. 2C).

Asthma and other allergic diseases are typified by the preferentialdifferentiation of naive T cells into TH2 cells. The presence TH2relevant cytokines and chemokines in Broncheoalveolar lavage fluid(BALF) was assessed. Toso−/− mice had a significant reduction in OVAinduced TH2 cytokines IL-4, IL-5 and IL-13 in BALF as assessed by ELISA(FIG. 3A-C). Consistent with the depressed eosinophil migration into thelung, Toso−/− mice had significantly reduced Eotaxin, an eosinophilattracting C—C chemokine, in the BALF (FIG. 3D). Bronchial smooth musclecells are known to produce eotaxin in response to TNFα. Since Toso isknown to be expressed in the lung, and Toso deficiency renders micerefractory to TNF, the inventors sought to address whether the effect ofToso on eotaxin levels was lung intrinsic. Cultured Toso−/− bronchialsmooth muscle cells had significantly reduced levels of Eotaxin producedin response to TNFα treatment (FIG. 3E). Taken together, these datasuggest that Toso deficiency impinges on OVA induced TH2 cytokine, andeotaxin levels in the lung.

A hallmark of allergic asthma is the overproduction of IgE. Antibodyproducing B cells are induced to produce IgE by TH2 cytokines such asIL-4 (Oettgen, H. C. and R. S. Geha, IgE regulation and roles in asthmapathogenesis. J Allergy Clin Immunol, 2001. 107(3): p. 429-40). OVAinduced total IgE and OVA specific IgE levels in the serum weresignificantly depressed in the Toso−/− mice (FIGS. 4A and B), whileOVA-specific IgG1 were similar between wild type and knockout (FIG. 4C).These data suggest that genetic ablation of Toso, or perhaps therapeuticblockade, could decrease the production of IgE.

The inflammatory events that typify allergic asthma culminate in airwayremodeling that cause restrictive airflow to the lung. Therefore,whether Toso deficiency affected OVA induced airway hyper-responsivenesswas assessed by measuring enhanced pause (Penh) in response toincreasing doses of methacholine using whole body plethysmography.Toso−/− mice were significantly protected from OVA induced airwayhyper-responsiveness as indicated by the significant reduction in Penhvalues compared to wild type controls (FIG. 5). These data aresuggestive of Toso blocking being an efficient strategy for mitigatingairway reactivity in allergic asthma.

Example 5 Toso Ablation Affects Dendritic Cell Activity

Toso ablation has wide-scale effects on the onset of allergic andinflammatory diseases. One possible non-limiting mechanism is that Tosoregulates these disease processes at a global level, perhaps through thefunction of dendritic cells (DCs) to present antigen to naïve T cells.

In order to test the functional relevance of Toso^(−/−) dendritic cellsin the onset of disease or in the activation of T cells, the inventorsdetermined whether the transfer of antigen loaded Toso−/− DCs eliciteddisease processes similar to wild type counterparts.

Wild type and Toso^(−/−) bone marrow derived DCs were made throughculture with GM-CSF (see Lutz, M. B., et al., An advanced culture methodfor generating large quantities of highly pure dendritic cells frommouse bone marrow. J Immunol Methods, 1999. 223(1): p. 77-92, which ishereby incorporated by reference in its entirety for all purposes and inparticular for all teaching regarding the culture of dendritic cells).Similar to the protocol of Lambrecht et al, the DCs in the presentexperiments were loaded with OVA. The antigen loaded DCs were instilledinto the trachea of C57BL6 animals (see Lambrecht, B. N., et al.,Myeloid dendritic cells induce Th2 responses to inhaled antigen, leadingto eosinophilic airway inflammation. J Clin Invest, 2000. 106(4): p.551-9, which is hereby incorporated by reference for all purposes and inparticular for all teachings related to dendritic cells). One weeklater, the animal were treated with aerosolled OVA as per the asthmastudies for three consecutive days. Strikingly, OVA loaded Toso^(−/−)dendritic cells were significantly reduced in their capacity to induceTH2 cytokine production into the BALF as compared to wild type controls(FIG. 7A). The 2d2 TCR transgenic strain contains T cells that arespecific for the MOG35-55 peptide used to elicit EAE (Bettelli, E., etal., Myelin oligodendrocyte glycoprotein-specific T cell receptortransgenic mice develop spontaneous autoimmune optic neuritis. J ExpMed, 2003. 197(9): p. 1073-81). T cells derived from 2d2 mice failed toproliferate when co-cultured with MOG35-55 loaded Toso−/− DCs (FIG. 7B).Mice expressing the major glycoprotein (GP) from lymphocytic choriomeningitis virus (LCMV) under control of the rat insulin promoter (RIP)develop diabetes as assessed by increased levels of serum glucose.Similarly, RIP-GP receiving GP peptide loaded DCs also develop disease.Toso−/− DCs show an impairment in their ability to elicit diseasecompared to wild type controls, and RIP-GP mice receiving peptide loadedToso−/− DCs survive significantly better than those mice receivingpeptide loaded wild type DCs (FIG. 7C). Taken together, these resultssuggest that Toso is necessary for the ability of Dendritic Cells toactivate T cells and induce disease.

Example 6 Administration of a Soluble Toso Protein Abrogates AsthmaDisease Metrics

To generate a soluble receptor, the extracellular domain of Toso fromhuman spleen cDNA library was amplified and then cloned in-frame with anFc domain derived from human IgG1 at the C-terminus intopFuse-hIgG1e3-Fc2 downstream of an IL2 signal sequence, allowing foroptimal secretion into the culture supernatant (FIG. 8A—signal sequenceindicated by a box, and Fc domain indicated with the underline). Inaddition, the Fc region contained several mutations(E233P/L234V/L235A/ΔG236/A327G/A330S/P331S) that have been shown toablate antibody dependent-, and complement dependent-cytotoxicity. Thesemutations can enhance the half-life of the protein and confer afavorable pharmacokinetic profile when administered in vivo. ThisToso-Fc construct, as well as an empty vector control, were transfectedinto 3×10⁸ 293 F cells in 400 ml of serum free media cells using theFreestyle expression system. Two days later, the supernatant wascollected and the cells were resuspended in a further 400 ml of media.After 2 more the days of culture, the second supernatant was collectedand combined with the first supernatant. The secretion of Toso-Fcsoluble receptor was confirmed using ELISA (FIG. 8B), purified byProtein G chromatography, and eluted with 100 mM Glycine pH 2.3. 1 mleluant fractions were collected into 60 μl of neutralization buffer (1 MTris pH 9.5) and confirmed by western blot with an antibody directed atthe Fc region (FIG. 8Ci) and Coomassie Blue staining (FIG. 8Cii).

Binding of the soluble receptor was tested on murine splenocytes.Approximately 106 cells were incubated with indicated amounts of Toso-Fcin 100 μl of FACS staining buffer (PBS+1% BSA+0.05% NaN₃) for 2 hours onice. Cells were washed in FACS buffer and incubated with a FITCconjugated anti-human FC g F(ab′)2 fragment for 30 minutes on ice, andacquired on a FACS Canto flow cytometer. Approximately 10% ofsplenocytes bound to the soluble receptor at 10 μg (FIG. 8D). A purifiedFc protein alone did not show significant binding to splenocytes,suggesting that Toso-Fc binding was specific. A further detailedanalysis indicated that Toso-Fc bound most significantly to CD11c+MHChimature dendritic cells or CD11c+B220+ plasmacytoid DCs in the spleen(FIG. 8E). These data illustrate that the Toso-Fc soluble receptor isfunctional, and that the ligand for Toso may be expressed on maturedendritic cells.

The therapeutic utility of Toso-Fc administration was assessed in amurine model of OVA induced asthma. Disease was elicited as describedabove with slight modifications. Female BALB/c mice were treated asdescribed except that 50 μg of Toso-Fc was administeredintraperitoneally for 3 consecutive days before aerosolled OVA challenge(FIG. 9A). Severity of disease was assessed by evaluating Th2 cytokinesin the BALF (FIG. 9B), cellularity in the BALF (FIG. 9C). Pretreatmentwith Toso-Fc significantly abrogated disease metrics, suggesting thatadministration of Toso-Fc may be a useful treatment paradigm in themanagement of asthma.

Example 7 Toso is a Potential Target for Treatment of Multiple Sclerosis

Experimental Autoimmune Encephalitis (EAE) is a useful mouse model forhuman MS. This system involves the antigen dependent activation of CD4+TH1/TH17 cells, and perivascular accumulation of monocytic cells leadingto demyelination, and hind limb paralysis. In the C57BL/6 background,EAE is deemed chronic progressive, as disease increases with time and iselicited through the injection of Myelin Oligodendrocyte Glycoproteinpeptide, MOG35-55, with Pertussis toxin in Complete Freund's Adjuvant.Disease severity is scored from 0 to 5 daily for approximately 1 month(where 0=no sign of disease, 1=limp tail or hind limb weakness but notboth, 2=limp tail and hind limb weakness, 3=partial hind limb paralysis,4=complete hind limb paralysis, 5=moribund state or death by EAE).

In the aforementioned MOG-induced model, Toso−/− mice were markedlyprotected from the onset of EAE compared to wild type controls (FIG. 6).These data suggest that blocking Toso function may represent a noveltreatment strategy for MS.

Example 8 Treatment with Toso-Fc Delays the Onset of EAE

This example demonstrates that treatment with Toso-Fc (SEQ ID NO: 5)delays the onset of EAE. As discussed above, the EAE mouse model is auseful model for human MS. For the experiments pictured in FIG. 23A,C57BL/6 mice were pretreated with Toso-Fc before immunization with thedisease-inducing MOG peptide. After immunization, mice were treated with50 μg Toso-Fc (or PBS for the control mice) intraperitoneally threetimes per week for 30 days. As shown in FIG. 23B, Toso-Fc delayed theprogression of EAE, showing a modifying effect of Toso-Fc in MultipleSclerosis.

Example 9 Toso Diminishes Innate Antibacterial Immune Response

This example demonstrates that granulocytes are activated early in Tosodeficient mice after bacterial infection and are essential for thecontrol of pathogens. Toso deficient granulocytes demonstrated enhancedexpression of CD11b and CD18 and displayed a lowered activationthreshold. In line with these results, Toso deficient granulocytesshowed reduced effector function at the sites of inflammation inperipheral tissue. As a result of the altered granulocyte function, Tosodeficient mice failed to clear systemic listeria infection leading tofast death of Toso deficient mice. Therefore, Toso influences activationthreshold and effector function of granulocytes, and thus criticallydiminishes innate anti bacterial immune response.

Materials and Methods

Mice:

Short sequences were obtained by sequencing the library isolated genomicDNA fragments using a series of oligomers derived from the mouse TosocDNA sequence. A 6.5 kbp fragment was isolated from the 5′ end usingBgIII restriction digestion enzyme and was used as a long arm for theknockout construct. A 650 bp short arm was produced by polymerase chainreaction using oligomers derived from the sequence of the 3′ end of thegene (5′ GTGAATACGTGAGCTTGGGCTACC 3′ SEQ ID NO: 26 and 5′CAAGTGATGGGGGATTACAGTGAA3′ SEQ ID NO: 27). The long and short arm were ligated oneither end of a Neomycin resistance cassette in the same orientation asthe Toso translation sense. The site specific insertion of this knockoutconstruct into embryonic stem (ES) cell genomic DNA was first screenedby polymerase chain reaction (PCR) using primers designed in the 3′ endof Neo and in the genomic DNA flanking region downstream of the 3′ endof the short arm. The mice were screened using three primers. A commonprimer (5′ TGTTTAATATGATGTGTCAGGCTG 3′ SEQ ID NO: 28) was located in theshort arm region and the two other primers were from either the 3′region of Neo (5′ AGGGCCAGCTCATTCCTCCCACTCAT 3′ SEQ ID NO: 29) or the 3′region of DNA that was excised by the knockout construct (5′AACTCTGCCCCTGCTCCTTCATTTCC 3′ SEQ ID NO: 30). In doing so the bandobtained from the rearranged allele was of 400 bp and that of the nativegene was 450 bp. D3 ES cells were electroporated with knockout constructand grown in the presence of 300 μg/ml G418. Positive ES clones werethen injected into E3.5 C57/BL6 derived blastocysts Chimeric off springswere screened for the presence of the rearranged allele and backcrossedto C57BL/6 background. CD11b^(−/−) mice were derived from Jackson on aC57BL/6 background.

Bone Marrow Chimeric Mice:

Mice were lethally irradiated with 1050 rad and reconstituted eitherwith 10⁷ CD45.1 WT bone marrow cells, or 10⁷ CD45.2 Toso^(−/−) bonemarrow cells or 5×10⁶ CD45.1 WT bone marrow cells plus 5×10⁶ CD45.2Toso^(−/−) bone marrow cells. All mice used in this study weremaintained on the C57BL/6 genetic background. All experiments wereperformed in single ventilated cages.

Listeria Infection:

Listeria was grown in heart infusion agar. If not differently indicated,mice were infected intravenously with 2×10⁴ CFU.

Granulocyte Activation and FACS Analysis after Cytokine Stimulation:

FACS staining and analysis were performed as described (Lang, P. A., etal. Aggravation of viral hepatitis by platelet-derived serotonin. NatMed 14, 756-761 (2008), which is hereby incorporated by reference in itsentirety for all purposes and in particular for all teachings related toFACS staining and analysis). Recombinant mouse TNF-α was from R+Dsystems, LPS was from Sigma and GM-CSF was obtained from X63O cellsupernatants. fMLP from Sigma was used at the indicated concentrations.For activation studies 10 μl blood was incubated in 100 μl mediumcontaining different cytokines in addition to anti Gr1 (eBiosciences),Dihydrorhodamin (Alexis) and CD11b (eBiosciences, if indicated). After30 minutes or 45 minutes of incubation (at 37° C. if not differentlyindicated) granulocytes were fixed and red cells were lysed usingerythrocyte lysis buffer (BD Biociences). For naïve expression of CD11a,CD11b and CD18 blood was fixed with 2% Formalin for 10 minutes, and thenstained with anti Gr1 and the according antibodies at 4° C. Tosoantibody was generated according to Nguyen, et al., (Blood, 2011, Jul.21, 118(3):598-608).

For priming studies granulocytes were incubated with different cytokinesfor 30 minutes followed by 15 minutes of fMLP incubation.

Histology:

Histological analysis was performed on snap frozen or formalin fixedtissue as described in Lang, K. S., et al. Immunoprivileged status ofthe liver is controlled by Toll-like receptor 3 signaling. J Clin Invest116, 2456-2463 (2006), which is hereby incorporated by reference in itsentirety for all purposes and in particular for all teachings related tohistological analysis.

MPO ELISA:

MPO ELISA was derived from Hycult biotech and performed according to themanufacturer's instructions.

mRNA Gene-Profiling by Quantitative RT-PCR:

RNA extraction and cDNA synthesis was performed using Trizol. Geneexpression analysis of was performed using Toso (Faim3) kitMm01302388_m1 from Applied Biosystems. For analysis, the expressionlevels of all target genes were normalized against 18sRNA (ΔCt). Geneexpression values are expressed as ΔCt.

Statistical Analysis:

Data are expressed as mean±S.E.M. Statistically significant differencesbetween two different groups were analyzed using Student's t-test. Ifnot differently mentioned unpaired two way. Analysis including severalgroups were performed using one-way ANOVA with additional Bonferroni orDunnett test. Statistically significant differences between experimentalgroups over multiple timepoints were calculated using two-way ANOVA(repeated measurements). p values <0.05 were considered as statisticallysignificant.

TOSO is Expressed on Granulocytes but is not Essential for LeukocyteDifferentiation

To analyze the functional relevance of Toso in vivo the inventorsgenerated a Toso deficient mouse as described in material and methods.Toso deficient mice were backcrossed to C57BL/6 background and thenanalyzed for expression of Toso RNA. RT-PCR analysis from Toso^(−/−)mice showed lack of Toso, suggesting that indeed Toso was not expressedin Toso^(−/−) mice (FIG. 12 a). Next Toso protein was analyzed onlymphocytes using a Toso specific antibody. It was found that naïve Bcells but not T cells expressed Toso (FIG. 12 b) in keeping withpublished literature. Next, the inventors analyzed expression of Toso ongranulocytes. The inventors found that granulocytes both in blood andspleen expressed Toso on the cell surface (FIG. 12 c). To analyze ifToso influenced lymphocyte or granulocyte development, the inventorsanalyzed these cell populations in peripheral blood of Toso^(−/−) mice.Toso^(−/−) mice did not show any striking difference in the bloodgranulocytes (FIG. 12 d). There was a slightly significant reduction ofblood lymphocyte numbers in Toso deficient mice which was attributed toa reduced B cell number (FIG. 12 d&e). The spleen size of Toso^(−/−)mice was normal and spleen lymphocytes showed a normal distribution(FIG. 12 f&g) suggesting that there is no major role of Toso on thedevelopment of immune cells.

3. Threshold for Activation is Reduced in Granulocytes of TOSO DeficientMice

Granulocytes from Toso^(−/−) and wildtype mice were activated with thegranulocyte activator N-Formylmethionyl-Lencyl-Phenylalanine (fMLP).fMLP activates the fMLP receptor on granulocytes, which mimics pathogencontact. In pre-activated granulocytes contact with fMLP leads to strongproduction of reactive oxygen species (ROS) and degranulation. Incontrast granulocytes which are not pre-activated by cytokines showlimited response to fMLP. Only 10% of wildtype granulocytes wereactivated upon treatment with fMLP when analyzed by production of ROSand degranulation (FIG. 13 a). In contrast, treatment with fMLPactivated 50% of Toso^(−/−) granulocytes (FIG. 13 a).

Next ROS production and degranulation of Toso^(−/−) granulocytes wasanalyzed following treatment with TNF-α, which usually only primesgranulocytes, but does not lead to ROS production. Treatment with TNF-αled to virtually absent ROS production in WT granulocytes (FIG. 13 b).In contrast, Toso^(−/−) granulocytes showed significant ROSproducing/de-granulated granulocytes after TNF-α treatment, suggestingthat Toso influences the threshold of granulocyte activation (FIG. 13b). Treatment with LPS and GM-CSF, which are also known to primegranulocytes did not activate ROS neither in wildtype nor Toso^(−/−)granulocytes (FIG. 13 c&d). These results show that Toso^(−/−)granulocytes had a lowered activation threshold for both fMLP and TNF-α.Co-treatment with GM-CSF or LPS together with fMLP increased thepercentage of ROS producing wildtype granulocytes by 10 fold in WTgranulocytes (FIG. 13 e). The percentage of ROS producing granulocyteswas also increased in either LPS or GM-CSF primed fMLP co-treatedToso^(−/−) granulocytes (FIG. 13 e). These data show that Toso^(−/−)granulocytes displayed a lowered activation threshold also in thesetting of granulocyte priming with GM-CSF or LPS. This suggests thatToso^(−/−) granulocytes had a reduced activation threshold in general.

Cellular stress can activate granulocytes, which might significantlycontribute to (auto)inflammatory disease. To analyze if Toso^(−/−)granulocytes are more prone to such activation, wildtype (WT) andToso^(−/−) granulocytes were incubated at different temperatures.Temperature reduction induced degranulation in 30% of Toso^(−/−)granulocytes, a condition where wildtype granulocytes did not show anyresponse (FIG. 13 f). The difference in the activation threshold couldbe explained by a difference in the life span between WT and Toso^(−/−)granulocytes. Granulocytes usually have a very limited life span whichis underlined by spontaneous apoptosis within 24 hours after in vitroculture. Granulocyte activation is usually coupled to prolonged lifespanand decreased apoptosis. However, there was no difference in spontaneousapoptosis between Toso^(−/−) and WT granulocytes (FIG. 17). Treatmentwith GM-CSF reduced apoptosis in granulocytes a process which did notdepend on Toso (FIG. 17). The data demonstrate that expression of Tosoprotein kept granulocytes in a resting non-activated state whenencountering inflammatory or stress signals.

Activation Threshold in Granulocytes is Regulated Intrinsically

Granulocytes derived from Toso^(−/−) mice had a lowered threshold fordifferentiation into an effector phenotype when compared to wildtypegranulocytes. Besides their intrinsic regulation, granulocyte activationis influenced by several extrinsic humoral factors (like: cytokines,antibodies, complement factors) as well as the activation state ofsurrounding cells such as B cells, T cells, endothelial cells orplatelets. To gain more insights into the mechanisms of loweredactivation threshold of granulocytes derived from Toso^(−/−) mice, themixed bone marrow chimeras were analyzed. C57BL/6 mice were irradiatedand then reconstituted with either WT, Toso^(−/−) or mixed WT/Toso^(−/−)(ratio 1:1) bone marrow. WT bone marrow could be tracked separately inchimeric mice by expression of the CD45.1 isoform (FIG. 14 a). Thismixed bone marrow chimeras allowed analysis of the activation thresholdof WT and Toso^(−/−) granulocyte derived from the same mouse. Observeddifferences in the activation threshold (especially those in the mixedchimeric mice) would suggest that Toso regulates activation ofgranulocytes intrinsically. Analysis of bone marrow chimeras 30 daysafter bone marrow transplantation showed that Toso^(−/−) granulocytesreconstituted in the same number as WT granulocytes suggesting againthat there was no major role of Toso in the development of granulocytes(FIG. 14 b). In the mixed chimeras total granulocytes (WT andToso^(−/−)) were comparable to the mice receiving bone marrow from asingle mouse strain (FIG. 14 b). There was not a striking advantage ofWT versus Toso^(−/−) granulocytes in the competitive setting of themixed bone marrow chimera (FIG. 14 b).

Next the activation threshold of WT versus Toso^(−/−) granulocytes inthe chimeric mice were analyzed. Treatment with fMLP activated moregranulocytes in mice which received bone marrow transplantation (FIG. 14a versus FIG. 13 a), which may, without being limited to this potentialmechanism, be due to different cytokine levels in mice receiving bonemarrow transplantation. Comparable to the data derived from theToso^(−/−) mice, the C57BL/6 mice, reconstituted with Toso^(−/−) bonemarrow, showed a lowered activation threshold (FIG. 14 c). This was alsoseen in the mixed bone marrow chimeras (FIG. 14 c). This suggests thatToso influenced the activation threshold of granulocytes intrinsically.

In order to determine if Toso in addition influenced the strength ofeffector function, the amount of ROS production of activated WT andactivated Toso^(−/−) granulocytes was determined. Activated WTgranulocytes showed significantly more ROS production than activatedToso^(−/−) granulocytes (FIG. 14 d). These data show that Toso enhancedthe activation threshold of granulocytes. Once granulocytes wereactivated, they showed enhanced effector function in the presence ofToso.

Impaired Control of Listeria in Toso Deficient Mice

The effect of the lack of Toso in granulocytes on bacterial control invivo was assessed. Infection with gram-positive Listeria monocytogenesis in particular strongly dependent on the fast activation ofgranulocytes. Infection with a sublethal dose of Listeria led toincreased death in Toso^(−/−) mice (FIG. 15 a), suggesting that Toso wasessential for the control of bacteria. Blood granulocytes in Toso^(−/−)mice showed enhanced degranulation and ROS production two days afterListeria inoculation (FIG. 15 b). This correlated with enhanced releaseof Myeloperoxidase in serum of Toso^(−/−) mice (FIG. 15 c).

One possibility was that Toso^(−/−) granulocytes were rapidly activatedin the blood, but may have failed to exert their full blown effectorfunction in the infected organ. To analyze this hypothesis, the infectedwildtype or Toso^(−/−) mice were infected with 2×10⁶ CFU of Listeria andhistology was analyzed after 20 hours. Granulomas were found in both WTand Toso^(−/−) mice the (FIG. 15 d), suggesting that Toso was notinvolved in granuloma formation. Staining for Listeria showed enhancedbacteria in the granuloma (FIG. 15 d), suggesting that the effectorfunction in the granuloma was effected by Toso.

Analysis of granulocytes one day after infection in the organ, showedreduced effector function (FIG. 15 e) and Listeria growth wassignificantly enhanced in liver and spleen of Toso^(−/−) mice (FIG. 15f). This was associated with spread of Listeria into the blood (FIG. 18)and finally also to the brain, which was probably the reason for deathof Listeria infected Toso^(−/−) mice (FIG. 15 f). These data show thatToso^(−/−) mice showed enhanced susceptibility to Listeria infection.This correlated with enhanced granulocytes activation in the blood,reduced effector function in the liver and spleen and spread of Listeriato the brain.

Toso Regulates Surface CD11b and CD18 Expression on Granulocytes

Toso deficient granulocytes display enhanced activation in the blood butreduced effector function of granulocytes in the tissue. This phenotypewould fit well to a different expression and/or different signaling ofintegrins. CD11a, CD11b and CD18 are important integrins for granulocyteactivation. Therefore the expression of CD11a, CD11b and CD18 wascompared in WT and Toso^(−/−) granulocytes. There was a significantlyenhanced expression of CD11 b and CD18 on naïve Toso deficientgranulocytes (FIG. 16 a). Expression of CD11a was not significantlydifferent between WT and Toso^(−/−) granulocytes (FIG. 16 a). Uponactivation with GM-CSF or LPS, CD11 b expression was up-regulated inboth WT and Toso^(−/−) granulocytes, however the difference ofexpression was decreasing with increasing LPS and GM-CSF concentrations(FIG. 16 b). LPS and GM-CSF reduced the activation threshold ofgranulocytes upon fMLP. Therefore the difference in the CD11b expressionbetween WT and Toso^(−/−) granulocytes could likely explain thedifferent activation threshold. To see if indeed CD11 b expression isinvolved in regulation of the activation threshold, the inventorsanalyzed the role of CD11 b in the system. As expected, lack of CD11 bled to reduced expression of CD18 (FIG. 16 c). Cd11b^(−/−) granulocytesshowed reduced activation and ROS production upon treatment with fMLPtogether with GM-CSF (FIG. 16 d). These data show that Toso^(−/−)granulocytes showed enhanced basal expression of CD11b, an integrinwhich influences the activation threshold of granulocytes. Thosefindings linked the enhanced CD11 b expression in Toso^(−/−)granulocytes with the reduced activation threshold in the absence ofToso.

Example 10 Toso Plays a Role in Glucose Tolerance and InsulinSensitivity

This example demonstrates that Toso plays a role in glucose toleranceand insulin sensitivity. FIG. 19 shows that Toso^(−/−) mice have normalfood intake and body weight after initiation of a high fat diet. For theexperiments in FIG. 19A, body weights of WT and Toso^(−/−) male micewere monitored since the initiation of high fat diet (starting at 5weeks of age). For FIG. 19B, food intake was measured at 14 weeks posthigh fat diet. Food intake was measured by housing animals singly, withdeterminations of the differences in food weight at the beginning andend of a 2-day period.

As shown in FIG. 20, Toso^(−/−) mice had enhanced glucose tolerance andinsulin sensitivity compared to wildtype mice after being on a high fatdiet for 10-13 weeks. Glucose Tolerance test was performed with wildtypeand Toso^(−/−) mice that were fed with high fat diet for 10-13 weekssince 5 weeks of age (note that similar results were obtained withfemale mice groups). The Insulin Tolerance test was performed withwildtype and Toso^(−/−) mice that were fed with high fat diet for 10-13weeks since 5 weeks of age.

Glucose tolerance tests were performed on overnight-fasted animalsbetween 9:30 and 11:30 a.m., utilizing a glucose dose of 1 g/kg of bodyweight injected intraperitoneally (i.p.) and measurements of glucoselevels at 0, 15, 30, 60, and 120 min after the injection.

Insulin tolerance tests were performed on overnight-fasted animalsbetween 9:30 and 11:30 a.m. or 4.5-hour fasted animals between 1:00-3p.m., utilizing human regular insulin (Humalog) at a dose of 0.5 U/kgbody weight, and blood glucose levels were measured at 0, 15, 30, 45,and 60 min after the injection.

In contrast to the mice fed a high fat diet, FIG. 21 A and FIG. 21 Bshow that Toso^(−/−) had similar glucose tolerance and insulinsensitivity to wildtype mice fed when both sets of mice were regularchow for 10-13 weeks. Glucose Tolerance test was performed with wildtypeand Toso^(−/−) male mice that were fed with regular chow (15-18 weeksold). Insulin Tolerance test was performed with wildtype and Toso^(−/−)male mice that were fed with regular chow (15-18 weeks old). FIG. 21 Aand FIG. 21 B suggest, without being limited by theory, that theprotective effect of Toso-deficiency requires a high fat diet. Glucosetolerance tests were performed on overnight-fasted animals between 9:30and 11:30 a.m., utilizing a glucose dose of 1 g/kg of body weightinjected intraperitoneally (i.p.) and measurements of glucose levels at0, 15, 30, 60, and 120 min after the injection. Insulin tolerance testswere performed on overnight-fasted animals between 9:30 and 11:30 a.m.or 4.5-hour fasted animals between 1:00-3 p.m., utilizing human regularinsulin (Humalog) at a dose of 0.5 U/kg body weight, and blood glucoselevels were measured at 0, 15, 30, 45, and 60 min after the injection.

Example 11 Treatment with Soluble Toso Improves Glucose Tolerance inWildtype Mice Fed with a High Fat Diet

This example demonstrates that treatment with a soluble Toso protein(SEQ ID NO: 5) improved glucose tolerance in wildtype mice fed with ahigh fat diet for 14 weeks. FIG. 22A shows data from wildtype male micebefore soluble Toso treatment. The glucose tolerance test was done withfasted wildtype mice fed with high fat diet (60% fat calories) for 14weeks since 5 weeks of age. FIG. 22B shows data from wildtype mice aftersoluble Toso treatment. The mice (high fat diet for 14 weeks) weretreated with soluble Toso-hIgG at a dose of 50 μg intraperitoneally onday 0, 2, 5, 7, 9, and 12. Glucose tolerance test was performed on day14 after overnight fasting. During the course of the soluble Tosoprotein treatment, the mice were continued with the high fat diet.Glucose tolerance tests were performed on overnight-fasted animalsbetween 9:30 and 11:30 a.m., utilizing a glucose dose of 1 g/kg of bodyweight injected intraperitoneally (i.p.) and measurements of glucoselevels at 0, 15, 30, 60, and 120 min after the injection.

Example 12 Generation of Stable Cell Lines Secreting Human and MouseToso-Fc

Toso-Fc encoding cDNAs were transfected into adherent HEK293T cellsusing Lipofectamine 2000. Single clones were selected in DMEM mediasupplemented with 10% Fetal Bovine serum and 0.2 mg/ml Zeocin. Highexpressing clones were expanded to 15 cm dishes, and split 1:2 atconfluence. At each split, stable transfectants were cultured in mediawith successively more serum free Freestyle media, and successively lessDMEM/FBS media to condition the cells for serum free growth. (e.g. —90%DMEM/FBS 10% Freestyle at split 1→100% Freestyle at split 10). Stabletransfectants conditioned to grow in serum free media grew in suspensionin an orbital shaker incubator (37° C., 8% CO2, 125 rpm) without a lossin viability.

The present specification provides a complete description of themethodologies, systems and/or structures and uses thereof in exampleaspects of the presently-described technology. Although various aspectsof this technology have been described above with a certain degree ofparticularity, or with reference to one or more individual aspects,those skilled in the art could make numerous alterations to thedisclosed aspects without departing from the spirit or scope of thetechnology hereof. Since many aspects can be made without departing fromthe spirit and scope of the presently described technology, theappropriate scope resides in the claims hereinafter appended. Otheraspects are therefore contemplated. Furthermore, it should be understoodthat any operations may be performed in any order, unless explicitlyclaimed otherwise or a specific order is inherently necessitated by theclaim language. It is intended that all matter contained in the abovedescription and shown in the accompanying drawings shall be interpretedas illustrative only of particular aspects and are not limiting to theembodiments shown. Unless otherwise clear from the context or expresslystated, any concentration values provided herein are generally given interms of admixture values or percentages without regard to anyconversion that occurs upon or following addition of the particularcomponent of the mixture. To the extent not already expresslyincorporated herein, all published references and patent documentsreferred to in this disclosure are incorporated herein by reference intheir entirety for all purposes. Changes in detail or structure may bemade without departing from the basic elements of the present technologyas defined in the following claims.

What is claimed:
 1. A method of treating multiple sclerosis in asubject, said method comprising administering to said subject acomposition comprising a therapeutically effective amount of a Tosocomposition, wherein said Toso composition comprises an extracellularToso domain, a signal sequence and an Fc domain.
 2. The method of claim1, wherein said soluble Toso protein comprises an amino acid sequenceaccording to SEQ ID NO:
 5. 3. The method of claim 1, wherein saidextracellular Toso domain is at least 95% identical to SEQ ID NO:
 8. 4.The method of claim 1, wherein said Fc domain comprises an amino acidsequence according to SEQ ID NO:
 3. 5. The method of claim 1, whereinsaid signal sequence is an IL-2 signal sequence.
 6. The method of claim1, wherein said Toso composition further comprises a linker between saidextracellular Toso domain and said Fc domain.
 7. A method for delayingonset of multiple sclerosis in a subject, said method comprisingadministering to said subject a composition comprising a therapeuticallyeffective amount of a Toso protein, wherein said Toso protein comprisesan amino acid sequence with at least 95% sequence identity to SEQ ID NO:5.
 8. A method for delaying onset of multiple sclerosis in a subject,said method comprising administering to said subject a compositioncomprising a therapeutically effective amount of a Toso composition,wherein said Toso composition comprises an extracellular Toso domain, anFc domain and a linker between said extracellular Toso domain and saidFc domain.
 9. The method of claim 8, wherein said extracellular Tosodomain comprises amino acids 1 to 104 of SEQ ID NO:
 8. 10. The method ofclaim 8, wherein said Fc domain comprises one or more mutations thatdiminish or ablate antibody-dependent and complement-dependentcytotoxicity.